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WO2023046110A1 - 联合表达CCR2b的工程化免疫细胞及其制备和应用 - Google Patents

联合表达CCR2b的工程化免疫细胞及其制备和应用 Download PDF

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WO2023046110A1
WO2023046110A1 PCT/CN2022/121035 CN2022121035W WO2023046110A1 WO 2023046110 A1 WO2023046110 A1 WO 2023046110A1 CN 2022121035 W CN2022121035 W CN 2022121035W WO 2023046110 A1 WO2023046110 A1 WO 2023046110A1
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cells
car
nkg2d
tumor
ccr2b
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PCT/CN2022/121035
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English (en)
French (fr)
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张曦
黄智宏
韩德平
燕妮
王调霞
朱晓娜
王宁
林伟雄
卢永
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广州百吉生物制药有限公司
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • A61K2039/5156—Animal cells expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/00—Fusion polypeptide
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    • C12N2510/00—Genetically modified cells

Definitions

  • the invention belongs to the field of tumor immunity and cell therapy, and in particular relates to an engineered immune cell jointly expressing CCR2b.
  • Cellular immunotherapy is an emerging tumor treatment mode with significant curative effect, and it is a new type of autoimmune anti-cancer treatment. It is a method of using biotechnology and biological agents to culture, modify and amplify immune cells collected from patients in vitro, and then infuse them back into the patient's body to stimulate and enhance the body's autoimmune function, so as to achieve the purpose of treating tumors.
  • T cells are an important class of lymphocytes involved in cellular immunity, and can specifically recognize and kill tumor cells through the signal transmission of antigen-presenting cells.
  • tumor cells can also be affected by the reduction or loss of antigenic epitopes, immunosuppression, and tumor heterogeneity (that is, the same malignant tumor exists in different patients or between tumor cells in different parts of the same patient from genotype to phenotype). Differences) and other ways to hinder the specific recognition of T cells, thereby evading the body's immune response.
  • Chimeric antigen receptor T cell (chimeric antigen receptor T cell, CAR-T) therapy was developed to address this problem.
  • the 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 co-stimulatory 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 reinfusion 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, which cannot fully activate the activity of T cells, and the therapeutic effect is not good.
  • the second-generation CAR-T introduced a co-stimulatory domain on the basis of 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 co-stimulatory domain to 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 CAR-T jointly expresses other auxiliary factors, such as the joint expression of IL-12 or the STAT3/5 binding domain in the IL-2R ⁇ cell, which helps to improve tumor killing Effects such as activity and safety.
  • CAR-T therapy has achieved satisfactory results in hematological tumors, there is still a lot of room for improvement in the therapeutic effect of CAR-T on solid tumors.
  • the reasons are: (1) Many solid tumors are difficult to be detected in the early stage, and have the characteristics of high malignancy, high recurrence rate, and poor prognosis. For example, 83% of colorectal cancer patients were in the middle and late stage when they were first diagnosed, and 44% of the patients had metastases to the liver, lung and other parts, and nearly half of the patients survived for less than 5 years; about 70% of ovarian cancer When patients are diagnosed, the cancer cells have already metastasized, and it is difficult to be cured by surgery, chemotherapy, and radiotherapy.
  • the recurrence rate after treatment is still as high as 70%; 90% of pancreatic cancer patients are diagnosed at an advanced stage, and the 5-year survival rate is only 7%.
  • tumor tissues often have an immunosuppressive microenvironment, which can hinder the migration and infiltration of CAR-T cells.
  • Many malignant solid tumors are also characterized by high heterogeneity, and a single target antigen often cannot achieve the best therapeutic effect, and there is a risk of recurrence. Therefore, CAR-T cell therapy for patients with malignant solid tumors such as colorectal cancer, ovarian cancer, and pancreatic cancer 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) with higher efficiency and 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) co-expressing CCR2b and its preparation method and application.
  • an engineered immune cell such as CAR-T cell
  • the first aspect of the present invention provides an engineered immune cell, the engineered immune cell is a T cell or NK cell, and the immune cell has the following characteristics:
  • the immune cell expresses a chimeric antigen receptor (chimeric antigen receptor, CAR), wherein the CAR targets surface markers of tumor cells, wherein the antigen-binding domain of the CAR includes the extracellular domain of NKG2D ; and
  • the immune cells express exogenous CCR2b 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-T cells chimeric antigen receptor T cells
  • CAR-NK cells Chimeric antigen receptor NK cells
  • the CCR2b 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 cell expresses a chimeric antigen receptor CAR that targets a surface marker of the tumor cell
  • CAR and CCR2b proteins are expressed in tandem.
  • CAR and CCR2b proteins are independently expressed.
  • 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 surface of the tumor.
  • the chimeric antigen receptor CAR is located on the cell membrane of the engineered immune cells.
  • the chimeric antigen receptor CAR is located on the cell membrane of the CAR-T cell.
  • the CCR2b protein is located on the cell membrane of the CAR-T cells.
  • L is nothing or a signal peptide sequence
  • NKG2D is an NKG2D extracellular domain or an active fragment thereof
  • H is none or hinge region
  • TM is the transmembrane domain
  • C is costimulatory signal domain
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ (including wild type, or mutants/modifiers thereof);
  • the "-" is a connecting peptide or a peptide bond.
  • said L is respectively selected from signal peptides of proteins in the following group: CD8, GM-CSF, CD4, CD28, CD137, or mutants/modifications thereof, or combinations thereof.
  • the H is selected from the hinge region of proteins in the following group: 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, or a mutation/modification thereof, or a combination thereof.
  • the C is selected from the co-stimulatory domains of proteins in the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10 , CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, or mutants/modifications thereof, or combinations thereof.
  • C is a co-stimulatory domain derived from 4-1BB.
  • amino acid sequence of the extracellular domain of NKG2D is as shown in SEQ ID NO:1 73-216 or as shown in SEQ ID No:4.
  • 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 is nothing or a signal peptide sequence
  • scFv is an antigen-binding domain
  • H is none or hinge region
  • TM is the transmembrane domain
  • C is costimulatory domain
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ or a mutation/modification thereof;
  • the "-" is a connecting peptide or a peptide bond.
  • the scFv is an antibody single-chain variable region sequence targeting a tumor antigen.
  • the scFv is an antibody single-chain variable region sequence targeting an antigen selected from the following group: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD271, GUCY2C, CD24, CD133 , CD44, CD166, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC3, BCMA, ErbB2, NKG2D ligands (ligands), LMP1, EpCAM, VEGFR-1, Lewis-Y, ROR1, Claudin18.2 , CEA or a combination thereof.
  • an antigen selected from the following group: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD271, GUCY2C, CD24, CD133 , CD44, CD166, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC3, BCMA, ErbB2, NKG2D ligands (ligands), LMP1, Ep
  • amino acid sequence of the NKG2D is shown in SEQ ID NO: 1, wherein the extracellular domain is 73-216.
  • amino acid sequence of the CCR2b protein is shown in SEQ ID NO:2.
  • first CAR shown in formula I and the second CAR shown in formula II can be combined into one to form a CAR shown in formula IIIa or IIIb:
  • L is nothing or a signal peptide sequence
  • NKG2D is an NKG2D extracellular domain or an active fragment thereof
  • scFv is an antigen-binding domain
  • H is none or hinge region
  • TM is the transmembrane domain
  • C is costimulatory domain
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ or a mutation/modification thereof;
  • the "-" is a connecting peptide or a peptide bond.
  • the 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:
  • step (B) including:
  • the step (B1) mentioned therein can be performed before, after, simultaneously or alternately with the step (B2).
  • a method for preparing the CAR-T cells of the present invention comprising the following steps:
  • step (B) in step (B), it includes (B1) introducing the first expression cassette expressing the CAR into the T cell; and (B2) introducing the second expression cassette expressing CCR2b into the T cell cells; wherein the step (B1) can be performed before, after, simultaneously, or alternately with the step (B2).
  • step (B) when the T cells to be modified in step (A) have already expressed CAR, then in step (B), including (B2) introducing a second expression cassette into the T cells.
  • the transcription directions of the first expression cassette and the second expression cassette are in the same direction ( ⁇ ), in opposite directions ( ⁇ ), and in opposite directions ( ⁇ ).
  • 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 a connecting peptide is further included between the first and second expression cassettes.
  • the connecting peptide is P2A.
  • 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 group consisting of 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.
  • the 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 formulation is a liquid formulation.
  • the dosage form of the preparation includes injection.
  • the concentration of the engineered immune cells (such as CAR-T cells) in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the fourth aspect of the present invention provides the use of the engineered immune cell according to the first aspect of the present invention for preparing a drug or preparation for preventing and/or treating cancer or tumor.
  • the use of the CAR-T cells according to the first aspect of the present invention is provided for the preparation of drugs or preparations for preventing and/or treating cancer or tumors.
  • the preparation contains CAR-T cells, and a pharmaceutically acceptable carrier, diluent or excipient.
  • 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, ovarian cancer, and pancreatic cancer.
  • the tumor is a tumor with high expression of NKG2D ligands (including any one of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or a combination thereof).
  • NKG2D ligands including any one of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or a combination thereof.
  • the tumor has high expression of NKG2D ligands (including any one of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or a combination thereof) and/or chemokines (Including tumors with high expression of any one of CCL2, CCL7, CCL8, CCL12, CCL13, and CCL16, or a combination thereof.
  • NKG2D ligands including any one of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or a combination thereof
  • chemokines Including tumors with high expression of any one of CCL2, CCL7, CCL8, CCL12, CCL13, and CCL16, or a combination thereof.
  • the fifth aspect of the present invention provides a kit for preparing the engineered immune cell described in the first aspect of the present invention, the kit includes a container, and in the container:
  • kits for preparing the engineered immune cell according to the first aspect of the present invention includes a container, and in the container:
  • first and second nucleic acid sequences are independent or linked.
  • the first and second nucleic acid sequences are located in the same or different containers.
  • the first and second nucleic acid sequences are located on the same or different vectors.
  • the first and second nucleic acid sequences are located in the same vector.
  • a third nucleic acid sequence is further 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.
  • the vector is a viral vector, preferably the viral vector contains the first and second nucleic acid sequences in tandem form.
  • FIG 1 shows the structures of CAR molecules of different generations.
  • Figure 2 shows the structure of the CAR molecule.
  • Figure 3 shows the expression rate of NKG2D CAR molecules detected by flow cytometry.
  • Figure 4 shows the expression rate of CEA CAR molecules detected by flow cytometry.
  • Figure 5 shows the expression rate of CCR2b detected by flow cytometry.
  • Figure 6 shows the expression rate of NKG2D ligands (MICA/MICB) in target cells detected by flow cytometry.
  • Figure 7 shows the expression rate of NKG2D ligands (ULBP-2/5/6) in target cells detected by flow cytometry.
  • Figure 8 shows the expression rate of NKG2D ligand (ULBP-3) in target cells detected by flow cytometry.
  • Figure 9 shows the expression rate of NKG2D ligand (ULBP-4) in target cells detected by flow cytometry.
  • Figure 10 shows the expression rate of CEA in target cells detected by flow cytometry.
  • Figure 11 shows the killing effect of NKG2D CAR-T cells detected by EuTDA method.
  • Figure 12 shows the killing effect of CEA CAR-T cells detected by EuTDA method.
  • Figure 13 shows the IFN- ⁇ release level of CAR-T cells detected by ELISA.
  • Figure 14 shows the principle and results of Transwell's detection of the chemotaxis and migration ability of CAR-T cells.
  • the inventors After extensive and in-depth research and extensive screening, the inventors firstly expressed specific CAR and CCR2b proteins, that is, CAR and CCR2b containing NKG2D extracellular domain (ED), in CAR-T cells.
  • the immune cells of the present invention can improve the sensitivity of CAR-T cells to chemokines such as CCL2 and CCL7 through CCR2b, and efficiently migrate to lesions of solid tumors such as colorectal cancer, ovarian cancer, and pancreatic cancer.
  • NKG2D CAR molecules can recognize multiple target antigens on the surface of malignant tumor cells such as colorectal cancer, ovarian cancer, and pancreatic cancer (including MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6) , to reduce the risk of decreased efficacy due to tumor heterogeneity or loss of target antigens.
  • malignant tumor cells such as colorectal cancer, ovarian cancer, and pancreatic cancer (including MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6)
  • MICA MICB
  • ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6 pancreatic cancer
  • studies have shown that NKG2D CAR-T can also target immunosuppressive cells and new blood vessels in the tumor microenvironment, helping T cells overcome the immunosuppressive tumor microenvironment and improve the effect of tumor treatment.
  • the present invention has been
  • the present invention takes CAR-T cells as an example to representatively describe the engineered immune cells of the present invention in detail.
  • the engineered immune cells of the present invention are not limited to the CAR-T cells described above, and the engineered immune cells of the present invention have the same or similar technical features and beneficial effects as the CAR-T cells described above.
  • immune cells express chimeric antigen receptor CAR
  • NK cells are equivalent to T cells (or T cells can be replaced by NK cells).
  • administration 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 intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal , spinal or other parenteral routes of administration, for example by injection or infusion.
  • the term "antibody” shall include, but not be limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds , or an antigen-binding portion thereof.
  • Each H chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three constant domains CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region comprises one constant domain, CL.
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • antigen-binding domain and “single-chain antibody fragment” all refer to a Fab fragment, Fab' fragment, F(ab') 2 fragment, or a single Fv fragment having antigen-binding activity.
  • Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant region, and the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding.
  • the antigen-binding domain is usually a scFv (single-chain variable fragment).
  • a single-chain antibody is preferably a sequence of one amino acid chain encoded by one nucleotide chain.
  • the scFv comprises an NKG2D extracellular domain or an active fragment thereof that specifically recognizes an antigen highly expressed by a tumor.
  • the immune cells of the present invention may also contain additional antibodies that specifically recognize antigens highly expressed in tumors, preferably single-chain antibodies or Fv antibodies.
  • a Chimeric antigen receptor includes an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes an optional signal peptide and a target-specific binding domain (also known as an antigen binding domain).
  • the intracellular domain includes the co-stimulatory domain and the CD3 ⁇ chain portion.
  • the extracellular segment can recognize a specific antigen, and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity and secretion of cytokines such as IL-2 and IFN- ⁇ etc., affecting tumor cells so that they do not grow, are induced to die, or are otherwise affected, and result in a reduction or elimination of the patient's tumor burden.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of the co-stimulatory molecule and the CD3zeta chain.
  • the antigen binding domain is fused to the intracellular domain of the combination of the 4-1BB signaling domain and the CD3 ⁇ signaling domain.
  • the CAR of the present invention targets NKG2D ligands and can specifically bind to MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • 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 NKG2D ligands, such as colorectal cancer, ovarian cancer, pancreatic cancer and the like.
  • CAR-T cells have the following advantages over other T-cell-based therapies: (1) The action process of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor antigen, it can target 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) using the patient's own 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 expression of NKG2D ligands, such as colorectal cancer, ovarian cancer, pancreatic cancer and the like.
  • NK cells are a major type of immune effector cells, which protect the body from virus infection and tumor cell invasion through non-antigen-specific pathways. NK cells through engineering (gene modification) may obtain new functions, including the ability to specifically recognize tumor antigens and have enhanced anti-tumor cytotoxicity.
  • CAR-NK cells Compared with autologous CAR-T cells, CAR-NK cells also have the following advantages, for example: (1) directly kill tumor cells by releasing perforin and granzymes, but have no killing effect on normal cells of the body; (2) they release A very small amount of cytokines reduces the risk of cytokine storm; (3) It is very easy to expand in vitro and develop into "off-the-shelf" products. Other than that, it is similar to CAR-T cell therapy.
  • NKG2D includes wild type or its mutant or its derivative form or its active fragment.
  • Preferred NKG2Ds are NKG2Ds from mammals such as humans and non-human primates.
  • accession number of the amino acid sequence of the human NKG2D protein is NP_031386, and the accession number of the nucleotide amino acid sequence is NM_007360.
  • the full-length amino acid sequence of human NKG2D is as follows:
  • positions 1-51 are intracellular domains; positions 52-72 are transmembrane regions; positions 73-216 are NKG2D extracellular domains (underlined).
  • Chemokines are a special class of cytokines, including more than 50 members. According to the structure, it is divided into four types: CC, CXC, CX3C and XC; chemokine receptors are correspondingly divided into four types: CCR, CXCR, CX3CR and XCR, with about 20 members.
  • the expressed chemokine receptor is CCR2b protein
  • the chemokines that can be combined include CCL2, CCL7, CCL8, CCL12, CCL13, CCL16 and the like.
  • accession number of the amino acid sequence of the CCR2b protein is NP_001116868.1, and the accession number of the nucleotide amino acid sequence is NM_001123396.4.
  • the specific sequence is as follows:
  • an "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, the first expression cassette comprises the nucleic acid sequence encoding the CAR.
  • the second expression cassette expresses exogenous CCR2b protein.
  • CCR2b protein can be expressed constitutively or inducibly.
  • the second expression cassette expresses the CCR2b protein; thus, when the CAR-T cells of the present invention are not exposed to the corresponding inducer, the second expression cassette does not Expresses CCR2b protein.
  • the first expression cassette and the second expression cassette further comprise a promoter and/or a terminator, respectively.
  • the promoter of the second expression cassette may be a constitutive or inducible promoter.
  • the present invention also provides a vector comprising the expression cassette of the present invention.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools for long-term gene transfer because they allow long-term, stable integration of the transgene in the genome of the cell and replication of the genome of the daughter cell.
  • 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 present invention can be connected downstream of the promoter through routine operations, and incorporated into an expression vector.
  • the vector can be integrated into the genome of eukaryotic cells and then replicated.
  • a typical cloning vector contains transcriptional and translational terminators, an initial sequence and a promoter useful for regulating the expression of the desired nucleic acid sequence.
  • the expression vectors of the 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, eg, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby 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 including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Particular vectors of interest include expression vectors, replication vectors, and the like.
  • expression vectors can be provided to cells in the form of viral vectors.
  • Viral vector technology is well known in the art and 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.
  • suitable vectors contain at least one origin of replication functional in the organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO01/96584; WO01/29058; and US Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo.
  • retroviral systems are known in the art.
  • lentiviral vectors are used.
  • Many DNA viral systems are known in the art.
  • an adenoviral vector is used.
  • Many adenoviral vectors are known in the art.
  • promoter elements can regulate the frequency of transcription initiation.
  • these elements are located in a region of 30-110 bp upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site.
  • the spacing between promoter elements is often flexible in order to preserve promoter function when an element is inverted or moved relative to another element.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased by 50 bp apart before activity begins to decline.
  • individual elements can 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 alpha (EF-1 alpha).
  • constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus (Epstein-Barr virus, EBV) immediate early promoter, Ruth's sarcoma virus promoter, and human gene promoters, such as but not limited to protein promoter, myosin promoter, heme promoter and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters.
  • inducible promoters are also contemplated as part of the invention.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence linked to the inducible promoter when desired, or turning off expression when not desired.
  • inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.
  • the expression vector introduced into the cells may also contain either or both of a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from the transfected or infected cell population by the viral vector.
  • selectable markers can be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker gene and the reporter gene may 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 easily introduced into host cells, eg, mammalian (eg, human T cells), bacterial, yeast or insect cells, by any method in the art.
  • expression vectors can be transferred into host cells by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, cationic complex transfection, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., 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 for 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, eg human, cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, eg, 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 lipid-based systems.
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and lipid-based systems.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle).
  • 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.
  • Lipid-associated nucleic acids can be encapsulated into the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached via linker molecules associated with both liposomes and oligonucleotides
  • linker molecules associated with both liposomes and oligonucleotides
  • entrapped in liposomes complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or Complexes with micelles, or otherwise associated with lipids.
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution.
  • Lipids are lipid substances, which may be naturally occurring or synthetic lipids.
  • lipids include fat droplets, which occur naturally in the cytoplasm as well as compounds comprising long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.
  • the vector is a lentiviral vector.
  • the present invention provides an engineered immune cell (such as CAR-T cell) according to the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • 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, etc.; 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, etc.
  • 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
  • preservatives e.g, aluminum hydroxide
  • the invention includes the therapeutic use of cells (eg, T cells) transduced with a vector (eg, a lentiviral vector) comprising an expression cassette of the invention.
  • a vector eg, a lentiviral vector
  • the transduced T cells can target the surface markers of tumor cells and express CCR2b 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 mammalian tumor cell populations or tissues, comprising the following steps: administering the CAR-T cells of the present invention to mammals.
  • the present invention includes a type of cell therapy, in which a patient's own T cells (or a heterologous donor) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient.
  • a patient's own T cells or a heterologous donor
  • the probability of graft-versus-host reaction is extremely low, and the antigen is recognized by T cells without MHC restriction.
  • a single CAR-T can treat all cancers that express that antigen.
  • CAR-T cells are able to replicate in vivo, resulting in 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 last for several months to several years.
  • the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-T cells can induce a specific immune response to tumor cells that overexpress the antigen recognized by the CAR antigen-binding domain.
  • the CAR-T cells of the present invention elicit a specific immune response against tumor cells with high expression of NKG2D ligands.
  • Treatable cancers include tumors that are not or substantially not vascularized, as well as vascularized tumors.
  • Cancer types treated with the CAR of the present invention include, but are not limited to: colorectal cancer, ovarian cancer, and pancreatic cancer.
  • the present 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 present invention.
  • the CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition with a diluent and/or in combination with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical compositions 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.
  • the pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or may be determined by clinical trials.
  • compositions of the invention to be administered can be determined by a physician, taking into account the patient (subject ) with individual differences in age, weight, tumor size, degree of infection or metastasis, and disease.
  • Pharmaceutical compositions comprising T cells described herein may be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably at a dose of 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 doses.
  • Cells can be administered using infusion techniques well known in immunotherapy (see, eg, 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 art 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.
  • Compositions of T cells can be injected directly into tumors, lymph nodes or sites 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., previously , simultaneously or subsequently) to the patient in a form of treatment including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or erfatizumab treatment for psoriasis patients or other treatments for PML patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or erfatizumab treatment for psoriasis patients or other treatments for PML patients.
  • the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents.
  • the cell composition of the invention is administered in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation, the use of chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • a subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • Dosages administered to a patient for the above treatments will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be implemented according to practice accepted in the art. Usually, 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, through intravenous infusion.
  • the present invention uses the CCR2b protein to enable the immune cells of the present invention to migrate to the tumor site more efficiently, thereby significantly improving the effect of inhibiting tumors and reducing toxic and side effects.
  • the present invention significantly improves the ability of CAR-T cells to migrate to places with high CCL2 concentration (such as lesion sites).
  • the antigen binding domain of the engineered immune cells of the present invention adopts the extracellular binding domain of NKG2D, which can recognize 8 kinds of targets on the cell surface of malignant tumors (such as colorectal cancer cells, ovarian cancer, pancreatic cancer, etc.) through NKG2D CAR molecules.
  • Spot antigens (MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6), reduce the risk of decreased efficacy due to tumor heterogeneity or loss of target antigens.
  • NKG2D CAR molecules also promoted the expression of exogenous CCR2b.
  • CEA CAR-T is used as the control of NKG2D CAR-T cells.
  • NKG2D CAR and CEA CAR molecules respectively include the following partial structures: human CD8 signal peptide [abbreviated as CD8 (SP)], human NKG2D extracellular domain [abbreviated as NKG2D (ED)], human anti-CEA single-chain antibody [abbreviated as CEAscFv (hMN- 14)], optimized human CD8 hinge region [abbreviated as CD8(hinge)] and human CD8 transmembrane domain [abbreviated as CD8(TM)], human 4-1BB intracellular domain [abbreviated as 4-1BB(ID)] , human CD3 ⁇ intracellular signal transduction domain [abbreviated as CD3 ⁇ (ID)], self-cleaving peptide P2A, human CCR2b.
  • CD8 signal peptide
  • ED human NKG2D extracellular domain
  • CEAscFv human anti-CEA single-chain
  • the second-generation NKG2D CAR molecule used as a control was named BN001;
  • the new-generation NKG2D CAR molecule co-expressing CCR2 is named BN003;
  • the second-generation CEA CAR molecule used as a control was named BC001, and the new-generation CEA CAR molecule co-expressing CCR2 was named BC006.
  • BN001 is composed of CD8 (SP), NKG2D (ED), CD8 (hinge), CD8 (TM), 4-1BB (ID) and CD3 ⁇ (ID) in series from its amino terminal to carboxyl terminal.
  • BN003 consists of CD8 (SP), NKG2D (ED), CD8 (hinge), CD8 (TM), 4-1BB (ID), CD3 ⁇ (ID), P2A, and CCR2b in series from its amino terminal to carboxyl terminal.
  • BC001 is composed of CD8 (SP), CEAscFv (hMN-14), CD8 (hinge), CD8 (TM), 4-1BB (ID) and CD3 ⁇ (ID) in series from its amino terminal to carboxyl terminal.
  • BC006 consists of CD8 (SP), CEAscFv (hMN-14), CD8 (hinge), CD8 (TM), 4-1BB (ID), CD3 ⁇ (ID), P2A, and CCR2b in series from its amino-terminus to carboxyl-terminus.
  • SEQ ID No: 3 amino acid sequence of human CD8 signal peptide
  • SEQ ID No: 4 amino acid sequence of human NKG2D extracellular domain
  • SEQ ID No:5 amino acid sequence of human anti-CEA single-chain antibody
  • SEQ ID No: 8 amino acid sequence of human 4-1BB intracellular domain
  • SEQ ID No:9 amino acid sequence of human CD3 ⁇ intracellular signal transduction domain
  • SEQ ID No:10 amino acid sequence of self-cleaving peptide P2A
  • Example 1 lentivirus preparation
  • nucleotide sequences of BN001, BN003, BC001, and BC006 were synthesized from the whole gene, and then connected to the lentiviral vector pCDH-EF1-MCS-T2A-copGFP plasmid by molecular cloning, so that it was in the human EF1 ⁇ promoter and Kozak sequence. expression under control.
  • lentiviral vector plasmids with lentiviral packaging plasmids pMD2.G, pRSV-Rev and pMDLg/pRRE with polyethyleneimine transfection reagent, and co-transfect 293T cells.
  • 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 the proportion of Jurkat cells successfully transduced by lentivirus was detected by flow cytometry.
  • the lentivirus infection rate of Jurkat cells was recorded as P, and the lentivirus titer was calculated by the following formula:
  • Lentivirus titer (TU/ml) P/V ⁇ 10 3 ⁇ 10 5
  • the titer of BN001 was 5.46 ⁇ 10 8 TU/ml
  • the titer of BN003 was 2.46 ⁇ 10 8 TU/ml
  • the titer of BC001 was 2.58 ⁇ 10 8 TU/ml
  • the titer of BC006 was 5.66 ⁇ 10 8 TU/ml.
  • the obtained T cells were washed, and the cell density was adjusted to 2 ⁇ 10 6 /ml.
  • Add lentivirus at MOI 1 ⁇ 10TU/cell for transduction, supplement 50ng/ml anti-CD3 antibody, 50ng/ml anti-CD28 antibody, and 200IU/ml recombinant IL-2 at the same time, and culture in a cell culture incubator ( The culture temperature was 37°C, and the carbon dioxide concentration was 5%). After 24 hours, the cell density was adjusted to 1.5-2 ⁇ 10 6 /ml, and 300 IU/ml of IL-2 was supplemented.
  • the cells On the 4th day after transduction, the cells were washed to remove residual lentiviral particles in the supernatant, and continued to be cultured in a cell incubator for 5 days (the culture temperature was 37°C, and the carbon dioxide concentration was 5%), during which the cell density was maintained at 1 ⁇ 2 ⁇ 10 6 /ml.
  • the obtained CAR-T cells followed the nomenclature of the corresponding CAR molecules, namely BN001, BN003, BC001 and BC006, and the T cells not transduced with lentivirus were named Ctrl T.
  • the cells of Ctrl T, BN001 and BN003 to be tested were washed twice with PBS, and resuspended with FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA). Add APC-labeled anti-human NKG2D antibody and PE-labeled anti-human CD3 antibody to the cell suspension to be tested according to the antibody instructions, and incubate at 4°C for 30 min. Using Ctrl T cells as a negative control, the expression rates of NKG2D CAR molecules in BN001 and BN003 cells were detected by flow cytometry. Analyzed by CytExpert software.
  • the Ctrl T, BC001 and BC006 cells to be tested were washed twice with PBS and resuspended with FACS buffer. Add APC-labeled CEA protein and PE-labeled anti-human CD3 antibody to the cell suspension to be tested according to the antibody instructions, and incubate at 4°C for 30min. Using Ctrl T cells not transfected with lentivirus as a negative control, the expression rates of CEA CAR molecules in BC001 and BC006 cells were detected by flow cytometry. Analyzed by CytExpert software.
  • the Ctrl T, BN001, BN003, BC001 and BC006 cells to be tested were washed twice with PBS and resuspended with FACS buffer. According to the antibody instructions, PE-labeled anti-human CCR2b antibody and APC-labeled anti-human CD3 antibody were added to the cell suspension to be detected, and incubated at 4°C for 30min. Using Ctrl T cells not transfected with lentivirus as a negative control, the expression rate of CCR2b in the above CAR-T cells was detected by flow cytometry. Analyzed by CytExpert software.
  • Tested colorectal cancer cell lines also known as target cells or target cell lines: HCT 116 (McCoy's 5a medium + 10% fetal bovine serum + 100U/ml penicillin + 100 ⁇ g/ml streptomycin), HT-29 (McCoy's 5a medium + 10% fetal bovine serum + 100U/ml penicillin + 100 ⁇ g/ml streptomycin), LoVo (F-12K medium + 10% fetal bovine serum + 100U/ml penicillin + 100 ⁇ g/ml streptomycin), SW480 (Leibovitz's L-15 medium + 10% fetal bovine serum + 100U/ml penicillin + 100 ⁇ g/ml streptomycin), T84 (DMEM/F-12 medium + 5% fetal bovine serum + 100U/ml penicillin + 100 ⁇ g /ml streptomycin), SK-OV-3 (McCoy's 5a medium+10% fetal bovine serum+100U/ml penicillin+
  • the above target cells were washed twice with PBS and resuspended with FACS buffer. Add the APC-labeled anti-human MICA/MICB antibody to each target cell suspension according to the antibody instructions, and incubate at 4°C for 30 min. The target cells incubated without adding antibodies were used as a negative control, and the MICA/MICB expression rate of the target cells was detected by flow cytometry. Analyzed by CytExpert software.
  • the MICA/MICB expression rate of HCT 166, HT-29, SW480 cells was between 96% and 100%
  • the MICA/MICB expression rate of T84 cells was about 56%
  • the MICA/MICB expression rate of LoVo cells The expression rate is about 7%
  • the expression rate of MICA/MICB in SK-OV-3 cells is about 1.8%.
  • the above target cells were washed twice with PBS and resuspended with FACS buffer. According to the antibody instructions, PE-labeled anti-human ULBP-2/5/6 antibodies were added to each target cell suspension, and incubated at 4°C for 30 min. The target cells incubated without adding antibodies were used as a negative control, and the expression rate of ULBP-2/5/6 in the target cells was detected by flow cytometry. Analyzed by CytExpert software.
  • the ULBP-2/5/6 expression rate of T84 cells is about 1.6%
  • the ULBP-2/5/6 expression rate of HCT116, HT-29, LoVo, SW480 cells is between 86-99%
  • SK - The expression rate of ULBP-2/5/6 in OV-3 cells was about 56.9%.
  • the above target cells were washed twice with PBS and resuspended with FACS buffer. According to the antibody instructions, PE-labeled anti-human ULBP-3 antibody was added to each target cell suspension, and incubated at 4°C for 30 min. The target cells incubated without adding antibodies were used as a negative control, and the ULBP-3 expression rate of the target cells was detected by flow cytometry. Analyzed by CytExpert software.
  • the ULBP-3 expression rate of HCT 166, HT-29, SW480 cells is between 59% and 95%
  • the ULBP-3 expression rate of LoVo cells is about 21%
  • the ULBP-3 expression rate of T84 cells The expression rate is lower than 3%
  • the expression rate of ULBP-3 in SK-OV-3 cells is about 33.1%.
  • the above target cells were washed twice with PBS and resuspended with FACS buffer. According to the antibody instructions, PE-labeled anti-human ULBP-4 antibody was added to each target cell suspension, and incubated at 4°C for 30 min. The target cells incubated without adding antibodies were used as a negative control, and the ULBP-4 expression rate of the target cells was detected by flow cytometry. Analyzed by CytExpert software.
  • the cells to be detected were washed twice with PBS and resuspended with FACS buffer. Add the anti-CEA primary antibody to the cell suspension to be tested according to the antibody instruction manual, and incubate at 4°C for 60 min. After washing with PBS, add FITC-labeled secondary antibody and incubate at 4°C for 60min. The target cells incubated only with FITC-labeled IgG were used as a negative control, and the CEA expression rate of the target cells was detected by flow cytometry. Analyzed by CytExpert software.
  • the CEA expression rate of LoVo cells is about 60%
  • the CEA expression rate of T84 cells is about 75%
  • the CEA expression rate of HCT 116, HT-29, SW480 is between 0.06% and 6%.
  • the expression rate of CEA in SK-OV-3 cells is about 0.01%.
  • HCT 116, HT-29, SW480, and SK-OV-3 cells were selected to detect the killing effect of NKG2D CAR-T cells, and LoVo and T84 were selected to detect the killing effect of CEA CAR-T cells Effect detection.
  • Wash target cells once with AIM-V medium Adjust the target cell density to 1 ⁇ 10 6 /ml, add DELFIA BATDA Reagent at 2 ⁇ l/ml, mix well, and incubate at 37°C for 30 minutes. After the target cells were washed three times with AIM-V medium, the target cells were seeded in a 96-well plate at a density of 1 ⁇ 10 4 /well.
  • T cells effect-to-target ratios are 2.5:1, 5:1, and 10:1
  • the culture temperature is 37° C., and the carbon dioxide concentration is 5%
  • centrifuge at 500 g for 5 min, and transfer 20 ⁇ l of the supernatant to a 96-well plate added with Europium solution (200 ⁇ l/well). After incubation at room temperature for 15 min, it was detected in a microplate reader.
  • Ctrl T, BN001, BN003, BC001, and BC006 were co-cultured with corresponding target cells in AIM-V medium without IL-2 (effect-to-target ratio 2.5:1). After 24 hours, dissolve the IFN- ⁇ standard product with ddH 2 O, place it at room temperature for 15-20 minutes to ensure full dissolution, and dilute the standard product according to the recommended gradient ratio. Aspirate the supernatant of the above co-cultured cells and dilute them 2-fold and 20-fold with ddH 2 O. The standard and experimental samples were added to the corresponding reaction wells, 100 ⁇ l per well.
  • the IFN- ⁇ release levels of BN001 to HCT 116 cells, HT-29 cells, SW480 cells and SK-OV-3 cells were 24525 ⁇ 453pg respectively /ml, 10066 ⁇ 644pg/ml, 5083 ⁇ 111pg/ml and 8309 ⁇ 979pg/ml;
  • the IFN- ⁇ release levels of BN003 on HCT 116 cells, HT-29 cells, SW480 cells and SK-OV-3 cells were 25502 ⁇ 1756pg/ml, 10524 ⁇ 312pg/ml, 7157 ⁇ 431pg/ml and 9055 ⁇ 241pg/ml;
  • the IFN- ⁇ release levels of BC001 on LoVo cells and T84 cells were 8611 ⁇ 480pg/ml and 10413 ⁇ 383pg/ml, respectively;
  • the IFN- ⁇ release levels of BC006 on LoVo cells and T84 cells were 4845 ⁇ 356p
  • the IFN- ⁇ of BN001, BN003, BC001 and BC006 cells were significantly higher than that of Ctrl T cells, indicating that the above-mentioned CAR-T cells can effectively release IFN- ⁇ after recognizing target cells;
  • the IFN- ⁇ release levels of HCT 116, HT-29 and SK-OV-3 cells were not significantly different, while the IFN- ⁇ release levels of BN003 to SW480 were significantly higher than those of BN001, indicating that in specific cases, co-expression of CCR2b can enhance NKG2D
  • the activity of CAR-T cells on the contrary, the IFN- ⁇ release level of LoVo and T84 cells by BC001 was significantly higher than that of BC006, indicating that the co-expression of CCR2b has a negative impact on the IFN- ⁇ release level of CEA CAR-T cells.
  • CCL2 protein 25 ng/ml
  • CAR-T cells 100 ⁇ l of CAR-T cells with a density of 4 ⁇ 10 5 /ml were added to the upper chamber of a Transwell plate with a pore size of 8 ⁇ m, and cultured in a carbon dioxide incubator (cultivation temperature was 37°C, carbon dioxide concentration was 5%). After 6 hours, the culture medium in the lower chamber was aspirated, the cells in it were mixed evenly, and the number of T cells migrating to the lower chamber was counted with a hemocytometer.
  • the chemokine concentration and culture time used in the Transwell experiment of the present invention are all equivalent to the prior art (experimental conditions are the same as patent CN111607006B), but the detected cell ratio (BN003 at 25ng/ml Under the condition of CCL2 induction for 6 hours, about 71.8% of the cells migrate) significantly higher than the data in the prior art (the KD-207 cells of this technology are under the condition of 25ng/ml CXCL2 induction for 6 hours, the highest about 40% cell migration), further illustrating that the NKG2D CAR-T cells co-expressing CCR2b of the present invention can efficiently undergo the desired directional migration.
  • mice Twenty-four B-NDG mice aged 6 to 8 weeks were taken for subcutaneous tumor drug efficacy experiment, and were randomly divided into 4 groups, 6 mice in each group, which were vehicle control group, Ctrl T control group, BN001 control group, and BN003 experimental group. .
  • the target cells in logarithmic growth phase and in good growth state were collected by trypsinization method, washed once with normal saline, and the cell density was adjusted to 2 ⁇ 10 7 /ml. 100 ⁇ l of cell suspension was subcutaneously injected into the right side of the B-NDG mice near the armpit, that is, each mouse was inoculated with 2 ⁇ 10 6 target cells, and the inoculation diary was regarded as day 0.
  • CAR-T cells (1 ⁇ 10 7 /monkey), Ctrl T cells (1 ⁇ 10 7 /monkey) and vehicle were respectively injected through the tail vein. (100 ⁇ l/only), the day of injection of the test substance was recorded as the 0th day of treatment.
  • the tumor size and mouse weight were measured 2 to 3 times a week, blood was collected on the 3rd, 10th, 28th, and 42nd day, and after adding EDTA for anticoagulation, qPCR was used to detect the presence of CAR-T cells in blood cells in mice.
  • IFN- ⁇ was detected by ELISA to monitor the release level of cytokines.
  • mice After 50 days of treatment, the mice were euthanized, and the tumor, heart, liver, spleen, lung, kidney, brain, ovary and other tissues were taken and weighed.
  • the tumor tissues of 2 mice in each group were stored in a refrigerator at 80°C for extraction DNA, to detect the infiltration of CAR-T cells in tumor tissues and their distribution in various organs; the tumor tissues of 2 mice in each group were fixed, and the morphology of tumor cells was detected by HE staining and immunohistochemistry. Detection of antigen expression in tissues.
  • mice injected with BN003 cells had a significant decrease in average tumor volume, by about 77%.
  • LTR sequences in tumor tissue were detected by qPCR to measure the infiltration of CAR-T cells in tumor tissue.
  • the results showed that the average background signal level of LTR in the tumor tissue of the mice in the vehicle control group was 30.5 copies/ ⁇ g DNA; the average background signal level of LTR in the tumor tissue of the mice in the Ctrl T control group was 25.2 copies/ ⁇ g DNA;
  • the average LTR content in the tumor tissue of the mice in the BN001 control group was 11852.0 copies/ ⁇ g DNA; the average LTR content in the tumor tissue of the mice in the BN003 experimental group was 47375.1 copies/ ⁇ g DNA.
  • the degree of infiltration of BN003 cells in tumor tissue was also significantly higher than that of BN001.
  • NKG2D also known as CD314
  • CD314 is an important activating receptor in the innate immune system, mainly expressed on the surface of natural killer cells, ⁇ T cells and CD8+T cells.
  • NKG2D as the antigen recognition domain of CAR molecules has many advantages. Unlike specific antibody CAR molecules targeting a single target, CAR molecules engineered based on NKG2D can recognize at least 8 different NKG2D ligands, including MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP- 4.
  • NKG2D ligands are all located on the surface of target cells. Therefore, compared with TCR-T, NKG2D CAR-T does not require the antigen presentation process of MHC molecules to directly recognize tumor cells. Importantly, NKG2D ligands are expressed at high levels in epithelial tumor cells such as colorectal cancer, ovarian cancer, pancreatic cancer, and leukemia, but are not expressed or expressed at very low levels in normal cells. ideal target. In addition, the surface of NKG2D CAR-T cells does not carry any foreign protein structures that may trigger the patient's immune response, thereby reducing the possibility of CAR-T cells being rejected by the patient's immune system.
  • Solid tumor cells can prevent the migration and infiltration of CAR-T cells into tumor tissue by secreting chemokines CXCL12 and CXCL5. In contrast, solid tumor cells secrete less chemokines that can promote CAR-T cell migration. These two factors make it difficult for CAR-T cells to reach solid tumor sites. Therefore, improving the specific recognition and sensitivity of CAR-T cells to tumor chemokines is one of the key factors affecting the therapeutic effect of CAR-T, and the combined expression of chemokine receptors in CAR-T cells is the solution to this problem. important method.
  • Chemokines are a special class of cytokines, including more than 50 members. According to the structure, it is divided into four types: CC, CXC, CX3C and XC; chemokine receptors are correspondingly divided into four types: CCR, CXCR, CX3CR and XCR, with about 20 members.
  • One of the main mechanisms of action of chemokines is to induce directional migration of immune cells by forming concentration gradients soluble or immobilized in the matrix, thereby regulating the infiltration of immune cells in tissues.
  • CAR-T technologies have used the combined expression of chemokine receptors to promote the rapid migration of CAR-T cells to cancer cells, thereby improving the tumor therapeutic effect of CAR-T cells.
  • the present invention not only efficiently solves the problem of targeting malignant tumor lesions, but also effectively overcomes the problem of malignant tumor heterogeneity through the specific combination of specific CAR molecules and specific chemokine receptors.
  • the present invention uses CCR2b as a co-expressed chemokine receptor to improve the ability of CAR-T cells to migrate to malignant tumor lesions, thereby improving the treatment efficiency.
  • CCL2 a chemokine mainly recognized by CCR2b, is abnormally highly expressed in various malignant tumors such as colorectal cancer, ovarian cancer, and pancreatic cancer, and its expression level is extremely low in normal tissue cells.
  • the affinity between CCR2b and CCL2 is extremely high (about 0.7nM, the smaller the IC 50 value, the higher the corresponding affinity and sensitivity), and the sensitivity is much higher than the combination of other chemokine receptors and chemokines (such as CCR2a/
  • the IC 50 value of the combination of CCL2 is about 5nM, and the C 50 value of the combination of CXCR3/CXCL11 is about 8.2nM).
  • the present invention uses NKG2D as the extracellular recognition domain of the CAR molecule, which can recognize various target antigens on the surface of tumor cells (including MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6), and can reduce
  • the risk of reduced efficacy due to tumor heterogeneity or antigen loss is more conducive to improving the therapeutic effect on malignant tumors such as colorectal cancer, ovarian cancer, and pancreatic cancer. Therefore, the present invention further improves the effectiveness of treatment, and improves the ability of immune cells such as CAR-T cells to fight against the high heterogeneity of malignant tumors.
  • NKG2D CAR-T will also target immunosuppressive cells and new blood vessels in the tumor microenvironment, helping immune cells such as T cells overcome the immunosuppressive tumor microenvironment and improve the effect of tumor treatment.

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Abstract

提供联合表达CCR2b的工程化免疫细胞，所述工程化免疫细胞为T细胞或NK细胞。将含有NKG2D胞外域的CAR分子和CCR2b联合表达于CAR-T细胞中，既能通过CCR2b提高CAR-T细胞对CCL2、CCL7等趋化因子的灵敏度，向结直肠癌、卵巢癌、胰腺癌等病灶高效迁移，提高治疗效率；同时又能通过NKG2D CAR分子识别结直肠癌、卵巢癌、胰腺癌等细胞表面的多种靶点抗原，降低因肿瘤异质性或靶点抗原丢失而导致疗效下降的风险，并提高肿瘤治疗的效果。

Description

联合表达CCR2b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CCR2b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。原因在于：(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。例如，83％的结直肠癌患者在首次确诊时已处于中晚期，且44％的患者已经出现了肝、肺等部位的转移，近半数患者生存期不到5年；约70％的卵巢癌患者在确诊时，癌细胞已经发生转移，难以通过手术、化疗、放疗手段治愈，治疗后的复发率仍高达70％以上；90％的胰腺癌患者被确诊时已属晚期，5年生存率仅7％。(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，单一靶点抗原往往不能达到最佳的治疗效果，且有复发的风险。因此，用于结直肠癌、卵巢癌、胰腺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CCR2b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NKG2D的胞外结构域；和
(b)所述的免疫细胞表达外源的CCR2b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CCR2b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细胞接触诱导剂时，所述CAR-T细胞诱导表达CCR2b蛋 白。
在另一优选例中，在所述的CAR细胞中，CAR和CCR2b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CCR2b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CCR2b蛋白定位于所述CAR-T细胞的细胞膜。
在另一优选例中，所述CAR的结构如式I所示：
L-NKG2D-H-TM-C-CD3ζ   (I)
式中，
L为无或信号肽序列；
NKG2D为NKG2D胞外结构域或其活性片段；
H为无或铰链区；
TM为跨膜结构域；
C为共刺激信号结构域；
CD3ζ为源于CD3ζ的胞浆信号传导序列(包括野生型、或其突变体/修饰体)；
所述“-”为连接肽或肽键。
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、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NKG2D的胞外结构域的氨基酸序列如SEQ ID NO:1第73-216位所示或如SEQ ID No:4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-scFv-H-TM-C-CD3ζ   (II)
式中，
L为无或信号肽序列；
scFv为抗原结合结构域；
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单链可变区序列。
在另一优选例中，所述scFv为靶向选自下组抗原的抗体单链可变区序列：CD19、CD20、CD22、CD123、CD47、CD138、CD33、CD30、CD271、GUCY2C、CD24、CD133、CD44、CD166、ABCB5、ALDH1、间皮素(mesothelin,MSLN)、EGFR、GPC3、BCMA、ErbB2、NKG2D配体(ligands)、LMP1、EpCAM、VEGFR-1、Lewis-Y、ROR1、Claudin18.2、CEA或其组合。
在另一优选例中，所述NKG2D的氨基酸序列如SEQ ID NO:1所示，其中胞外结构域为第73-216位。
在另一优选例中，所述CCR2b蛋白的氨基酸序列如SEQ ID NO:2所示。
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-NKG2D-scFv-H-TM-C-CD3ζ   (IIIa)
L-scFv-NKG2D-H-TM-C-CD3ζ   (IIIb)
式中，
L为无或信号肽序列；
NKG2D为NKG2D胞外结构域或其活性片段；
scFv为抗原结合结构域；
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CCR2b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NKG2D的胞外结构域。
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CCR2b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CCR2b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CCR2b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(→←)、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。
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1×10 3-1×10 8个细胞/ml，较佳地1×10 4-1×10 7个细胞/ml。
本发明的第四方面，提供了如本发明第一方面所述的工程化免疫细胞的用途，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、卵巢癌、胰腺癌。
在另一优选例中，所述肿瘤为NKG2D配体(包括MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6中的任意一种，或其组合)高表达的肿瘤。
在另一优选例中，所述肿瘤为NKG2D配体(包括MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6中的任意一种，或其组合)高表达和/或趋化因子(包括CCL2、CCL7、CCL8、CCL12、CCL13、CCL16中的任意一种，或其组合)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NKG2D的胞外结构域；和
(2)第二核酸序列，所述第二核酸序列含有用于联合表达CCR2b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CCR2b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。
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NKG2D CAR分子的表达率。
图4显示了流式检测CEA CAR分子的表达率。
图5显示了流式检测CCR2b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-2/5/6)的表达率。
图8显示了流式检测靶细胞中NKG2D配体(ULBP-3)的表达率。
图9显示了流式检测靶细胞中NKG2D配体(ULBP-4)的表达率。
图10显示了流式检测靶细胞中CEA的表达率。
图11显示了EuTDA法检测NKG2D CAR-T细胞的杀伤效果。
图12显示了EuTDA法检测CEA CAR-T细胞的杀伤效果。
图13显示了ELISA检测CAR-T细胞的IFN-γ释放水平。
图14显示了Transwell检测CAR-T细胞的趋化迁移能力的原理及结果。
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CCR2b蛋白，即将含NKG2D胞外结构域(ED)的CAR和CCR2b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CCR2b提高CAR-T细胞对CCL2、CCL7等趋化因子的灵敏度，向结直肠癌、卵巢癌、胰腺癌等实体瘤的病灶高效迁移，提高治疗效率；同时又能通过NKG2D CAR分子识别结直肠癌、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)，降低因肿瘤异质性或靶点抗原丢失而导致疗效下降的风险。同时，有研究表明NKG2D CAR-T也可靶向肿瘤微环境中的免疫抑制细胞和新生血管，有助于T细胞克服免疫抑制的肿瘤微环境，提高肿瘤治疗的效果。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，其特异性结合抗原并包含通过二硫键互连的至少两条重(H)链和两条轻(L)链，或其抗原结合部分。每条H链包含重链可变区(本文缩写为VH)和重链恒定区。重链恒定区包含三个恒定结构域CH1、CH2和CH3。每条轻链包含轻链可变区(本文缩写为VL)和轻链恒定区。轻链恒定区包含一个恒定结构域CL。VH和VL区可以进一步细分为称为互补决定区(CDR)的高变区，其散布有更保守的称为框架区(FR)的区域。每个VH和 VL包含三个CDR和四个FR，从氨基末端到羧基末端按照以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。
抗原结合结构域
如本文所用，“抗原结合结构域”“单链抗体片段”均指具有抗原结合活性的Fab片段，Fab'片段，F(ab') 2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。抗原结合结构域通常是scFv(single-chain variable fragment)。单链抗体优选是由一条核苷酸链编码的一条氨基酸链序列。
在本发明中，所述scFv包含特异性识别肿瘤高表达的抗原的NKG2D胞外结构域或其活性片段。
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，较佳地为单链抗体或Fv抗体。
嵌合抗原受体(CAR)
如本文所用，嵌合免疫抗原受体(Chimeric antigen receptor，CAR)包括细胞外结构域、任选的铰链区、跨膜结构域、和细胞内结构域。胞外结构域包括任选的信号肽和靶点特异性结合结构域(也称为抗原结合结构域)。细胞内结构域包括共刺激结构域和CD3ζ链部分。CAR在T细胞中表达时，胞外段可识别一个特异的抗原，随后通过胞内结构域转导该信号，引起细胞的活化增殖、细胞溶解毒性和分泌细胞因子如IL-2和IFN-γ等，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和CD3ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与4-1BB信号传导结构域和CD3ζ信号结构域组合的细胞内结构域融合。
在一个实施方式中，本发明CAR靶向NKG2D配体，能与MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6特异性结合。
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NKG2D配体高表达的肿瘤，如结直肠癌、卵巢癌、胰腺癌等。
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NKG2D配体高表达的肿瘤，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。
NKG2D
在本发明中，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的全长氨基酸序列如下所示：
Figure PCTCN2022121035-appb-000001
其中，第1-51位是胞内结构域；第52-72位是跨膜区；第73-216位是NKG2D胞外结构域(下划线部分)。
趋化因子
趋化因子是一类特殊的细胞因子，包含50多个成员。根据结构分为CC、CXC、CX3C和XC四类；趋化因子受体则相应分为CCR、CXCR、CX3CR和XCR4种，约有20个成员。
在本发明的工程化免疫细胞中，表达的趋化因子受体是CCR2b蛋白，可以结 合的趋化因子包括CCL2、CCL7、CCL8、CCL12、CCL13、CCL16等。
CCR2b蛋白的氨基酸序列的登录号为NP_001116868.1，核苷酸氨基酸序列的登录号为NM_001123396.4。具体序列如下所示：
氨基酸序列：
Figure PCTCN2022121035-appb-000002
核苷酸序列：
Figure PCTCN2022121035-appb-000003
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CCR2b蛋白。
在本发明中，CCR2b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细胞被相应诱导剂激活时，第二表达盒表达CCR2b蛋白；这样，在本发明CAR-T细胞在未接触相应诱导剂时，第二表达盒不表达CCR2b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。
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1×10 3-1×10 8个细胞/ml，更优地1×10 4-1×10 7个细胞/ml。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明包括含本发明表达盒的载体(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CCR2b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NKG2D配体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、卵巢癌和胰腺癌。
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10 4至10 9个细胞/kg体重的剂量，优选10 5至10 7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1×10 5个至1×10 10个本发明经修饰的T细胞，通过例如静脉回输的方式，施用于患者。
本发明的主要优点
(1)本发明利用CCR2b蛋白使得本发明的免疫细胞更高效地迁移到肿瘤部位，从而显著提高抑制肿瘤的效果，并可减少毒副作用。实验表明，本发明显著提高了CAR-T细胞向CCL2浓度高处迁移(如病灶部位)的能力。
(2)本发明的工程化免疫细胞的抗原结合域采用NKG2D的胞外结合域，可通过NKG2D CAR分子识别恶性肿瘤(如结直肠癌细胞、卵巢癌、胰腺癌等)细胞表面的8种靶点抗原(MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6)，降低因肿瘤异质性或靶点抗原丢失而导致疗效下降的风险。
(3)外源CCR2b蛋白的表达，意外地促进了NKG2D CAR分子在免疫细胞中的表达率。
(4)NKG2D CAR分子的表达也促进了外源CCR2b的表达。
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CEA CAR-T作为NKG2D CAR-T细胞的对照。NKG2D CAR 和CEA CAR分子分别包括以下部分结构：人CD8信号肽[简称CD8(SP)]、人NKG2D胞外结构域[简称NKG2D(ED)]、人抗CEA单链抗体[简称CEAscFv(hMN-14)]、优化了的人CD8铰链区[简称CD8(hinge)]和人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、人CCR2b。
作为对照的第二代NKG2D CAR分子命名为BN001；
联合表达CCR2的新一代NKG2D CAR分子命名为BN003；
作为对照的第二代CEA CAR分子命名为BC001，联合表达CCR2的新一代CEA CAR分子命名为BC006。
所述CAR分子的具体结构如图2所示，具体如下：
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。
BN003从其氨基端至羧基端由CD8(SP)、NKG2D(ED)、CD8(hinge)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)、P2A、CCR2b依次串联组成。
BC001从其氨基端至羧基端由CD8(SP)、CEAscFv(hMN-14)、CD8(hinge)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)依次串联组成。
BC006从其氨基端至羧基端由CD8(SP)、CEAscFv(hMN-14)、CD8(hinge)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)、P2A、CCR2b依次串联组成。
氨基酸序列
SEQ ID NO:1(NKG2D氨基酸序列)
Figure PCTCN2022121035-appb-000004
SEQ ID NO:2(CCR2b氨基酸序列)
Figure PCTCN2022121035-appb-000005
SEQ ID No:3(人CD8信号肽氨基酸序列)
Figure PCTCN2022121035-appb-000006
SEQ ID No:4(人NKG2D胞外结构域氨基酸序列)
Figure PCTCN2022121035-appb-000007
SEQ ID No:5(人抗CEA单链抗体氨基酸序列)
Figure PCTCN2022121035-appb-000008
SEQ ID No:6(优化了的人CD8铰链区氨基酸序列)
Figure PCTCN2022121035-appb-000009
SEQ ID No:7(人CD8跨膜结构域氨基酸序列)
Figure PCTCN2022121035-appb-000010
SEQ ID No:8(人4-1BB胞内结构域氨基酸序列)
Figure PCTCN2022121035-appb-000011
SEQ ID No:9(人CD3ζ胞内信号转导结构域氨基酸序列)
Figure PCTCN2022121035-appb-000012
SEQ ID No:10(自剪切肽P2A氨基酸序列)
Figure PCTCN2022121035-appb-000013
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BN001、BN003、BC001及BC006的核苷酸序列，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1×10 5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1.5-ml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×10 3×10 5
结果：BN001滴度为5.46×10 8TU/ml，BN003滴度为2.46×10 8TU/ml，BC001滴度为2.58×10 8TU/ml，BC006滴度为5.66×10 8TU/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2×10 6/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2×10 6/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1.5～2×10 6/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1～2×10 6/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BN001、BN003、BC001和BC006，未用慢病毒转导的T细胞命名为Ctrl T。
2.3 NKG2D CAR分子的表达检测
用PBS清洗待检测的Ctrl T、BN001和BN003细胞两次，并用FACS缓冲液(含0.1％叠氮化钠和0.4％BSA的PBS)重悬。按照抗体说明书将APC标记的抗人NKG2D抗体和PE标记的抗人CD3抗体加入待检测细胞悬液中，4℃孵育30min。以Ctrl T细胞为阴性对照，用流式细胞仪检测BN001和BN003细胞的NKG2D CAR分子的表达率。采用CytExpert软件分析。
结果如图3所示，BN001细胞的NKG2D CAR分子表达率约为87.2％，BN003细胞的NKG2D CAR分子表达率约为92.2％。出乎意料地发现，联合表达CCR2b使NKG2D CAR分子的表达率上升(提高约5.7％)。这提示，联合表达CCR2b+NKG2D的BN003的杀伤力有提高。
2.4 CEA CAR分子的表达检测
用PBS清洗待检测的Ctrl T、BC001和BC006细胞两次，并用FACS缓冲液重悬。按照抗体说明书将APC标记的CEA蛋白和PE标记的抗人CD3抗体加入待检测细胞悬液中，4℃孵育30min。以未用慢病毒转染的Ctrl T细胞为阴性对照，用流式细胞仪检测BC001和BC006细胞的CEA CAR分子的表达率。采用CytExpert软件分析。
结果如图4所示，BC001细胞的CEA CAR分子表达率约为71.5％，BC006细胞的CEA CAR分子表达率约为36.1％。联合表达CCR2b使CEA CAR分子的表达率下降(下降幅度为约50.5％)。这提示，联合表达CCR2b的BC006的杀伤力会下降。
2.5 CCR2b的表达检测
用PBS清洗待检测的Ctrl T、BN001、BN003、BC001和BC006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CCR2b抗体和APC标记的抗人CD3抗体加入待检测细胞悬液中，4℃孵育30min。以未用慢病毒转染的Ctrl T细胞为阴性对照，用流式细胞仪检测上述CAR-T细胞的CCR2b表达率。采用CytExpert软件分析。
结果如图5所示，BC006细胞的CCR2b表达率约为26.67％，BN003细胞的CCR2b表达率约为55.4％，说明与CEA联合表达时CCR2b的表达水平会下降。
实施例3靶细胞检测
3.1靶细胞培养条件
供试结直肠癌细胞系(又称靶细胞或靶细胞系)：HCT 116(McCoy's 5a培养基+10％胎牛血清+100U/ml青霉素+100μg/ml链霉素)，HT-29(McCoy's 5a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)，T84(DMEM/F-12培养基+5％胎牛血清+100U/ml青霉素+100μg/ml链霉素)，SK-OV-3(McCoy's 5a培养基+10％胎牛血清+100U/ml青霉素+100μg/ml链霉素)。
3.2 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，HCT 166、HT-29、SW480细胞的MICA/MICB表达率在96％～100％之间，T84细胞的MICA/MICB表达率约为56％，LoVo细胞的MICA/MICB表达率约为7％，SK-OV-3细胞的MICA/MICB表达率约为1.8％。
3.3 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ULBP-2/5/6表达率。采用CytExpert软件分析。
结果如图7所示，T84细胞的ULBP-2/5/6约为1.6％，HCT116、HT-29、LoVo、SW480细胞的ULBP-2/5/6表达率在86～99％间，SK-OV-3细胞的ULBP-2/5/6表达率约为56.9％。
3.4 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ULBP-3表达率。采用CytExpert软件分析。
结果如图8所示，HCT 166、HT-29、SW480细胞的ULBP-3表达率在59％～95％之间，LoVo细胞的ULBP-3表达率约为21％，T84细胞的ULBP-3表达率低于3％，SK-OV-3细胞的ULBP-3表达率约为33.1％。
3.5 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ULBP-4表达率。采用CytExpert软件分析。
结果如图9所示，HCT 116、HT29、LoVo、SW480的ULBP-4表达率均在82.2％以上，T84的ULBP-4表达率约为2.0％，SK-OV-3细胞的ULBP-4表达率约为71.6％。
3.6 CEA的表达检测
用PBS清洗待检测细胞两次，并用FACS缓冲液重悬。按照抗体说明书将抗CEA一抗加入待检测细胞悬液中，4℃孵育60min。用PBS清洗后再加入FITC标记的二抗，4℃孵育60min。以只用FITC标记的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CEA表达率。采用CytExpert软件分析。
结果如图10所示，LoVo细胞的CEA表达率约为60％，T84细胞的CEA表达率约为75％，而HCT 116、HT-29、SW480的CEA表达率在0.06％～6％间，SK-OV-3细胞的CEA表达率约为0.01％。
实施例4 CAR-T细胞的体外功能
4.1 EuTDA法杀伤效果检测
根据上述靶点抗原的流式检测结果，选用HCT 116、HT-29、SW480、SK-OV-3细胞进行NKG2D CAR-T细胞的杀伤效果检测，选用LoVo、T84进行CEA CAR-T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1×10 6/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1×10 4/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500g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。
结果如图11所示，在靶效比为2.5:1、5:1和10:1的条件下，BN001对HCT 116细胞的杀伤率分别为45％±2％、81％±4％、91％±1％，对HT-29细胞的杀伤率分别为49％±5％、87％±5％、90％±6％，对SW480的杀伤率分别为55％±9％、70％±9％、 66％±1％，对SK-OV-3的杀伤率分别为14％±1％、25％±1％、42％±1％；在靶效比为2.5:1、5:1和10:1的条件下，BN003对HCT 116细胞的杀伤率分别为50％±1％、71％±5％、84％±6％，对HT-29细胞的杀伤率分别为43％±8％、78％±8％、90％±6％，对SW480的杀伤率分别为43％±3％、61％±5％、67％±3％，对SK-OV-3的杀伤率分别为14％±1％、25％±1％、46％±2％。经统计学分析可知，BN001和BN003对相应靶细胞的杀伤效果没有统计学差异，且都显著高于Ctrl T细胞，说明联合表达CCR2b不会对NKG2D CAR-T细胞的杀伤效果产生负面影响(在共培养时，由于CAR-T细胞无需通过趋化迁移即可与靶细胞直接接触，所以在体外检测实验中，即使未观察到联合表达CCR2b对CAR-T细胞杀伤效果的提升作用，也属于正常情况)。
另外，如图12所示，在靶效比为2.5:1、5:1和10:1的条件下，BC001对LoVo细胞的杀伤率分别为56％±2％、89％±1％、96％±4％，对T84细胞的杀伤率分别为26％±1％、49％±6％、77％±3％；在靶效比为2.5:1、5:1和10:1的条件下，BC006对LoVo细胞的杀伤率分别为49％±6％、64％±8％、86％±12％，对T84细胞的杀伤率分别为28％±1％、46％±2％、75％±2％。经统计学分析可知，BC001和BC006细胞对T84细胞的杀伤效果没有统计学差异，但BC006对LoVo细胞的杀伤效果显著低于BC001，说明在部分情况下，联合表达CCR2b会导致CEA CAR-T细胞的杀伤效果下降。
4.2 IFN-γ分泌检测
将Ctrl T、BN001、BN003、BC001和BC006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ddH 2O溶解IFN-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ddH 2O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。
结果如图13所示，在靶效比为2.5:1的条件下，BN001对HCT 116细胞、HT-29细胞、SW480细胞和SK-OV-3细胞的IFN-γ释放水平分别为24525±453pg/ml、10066±644pg/ml、5083±111pg/ml和8309±979pg/ml；BN003对HCT 116细胞、HT-29细胞、SW480细胞和SK-OV-3细胞的IFN-γ释放水平分别为25502±1756pg/ml、10524±312pg/ml、7157±431pg/ml和9055±241pg/ml；BC001对LoVo细胞和T84细胞的IFN-γ释放水平分别为8611±480pg/ml和10413±383pg/ml；BC006 对LoVo细胞和T84细胞的IFN-γ释放水平分别为4845±356pg/ml和6279±382pg/ml。经统计学分析可知，BN001、BN003、BC001和BC006细胞的IFN-γ都显著高于Ctrl T细胞，说明上述CAR-T细胞可以在识别靶细胞后有效释放IFN-γ；其中，BN001和BN003对HCT 116、HT-29和SK-OV-3细胞的IFN-γ释放水平没有显著差异，而BN003对SW480的IFN-γ释放水平显著高于BN001，说明在特定情况下，联合表达CCR2b可以增强NKG2D CAR-T细胞的活性；相反，BC001对LoVo和T84细胞的IFN-γ释放水平都显著高于BC006，说明联合表达CCR2b对CEA CAR-T细胞的IFN-γ释放水平有负面影响。
4.3 Transwell趋化迁移检测
在Transwell 24孔平板的下室加入600μl以完全培养基稀释的CCL2蛋白(25ng/ml)。在孔径为8μm的Transwell平板上室分别加入100μl密度为4×10 5/ml的CAR-T细胞，并置于二氧化碳培养箱培养(培养温度为37℃，二氧化碳浓度为5％)。6h后，吸取下室的培养基，混匀其中的细胞后用血球计数板计算迁移到下室的T细胞数目。
结果如图14所示，当下室未加入CCL2蛋白时，BN001、BN003、BC001和BC006迁移到下室的细胞数分别约为2.14×10 5个、1.99×10 5个、9.90×10 4个、1.20×10 5个；当下室加入25ng/ml的CCL2蛋白时，BN001、BN003、BC001和BC006迁移到下室的细胞数分别约为2.18×10 5个、2.87×10 5个、8.61×10 4个、1.34×10 5个。BN003迁移到下室的细胞数目显著高于作为对照的BN001细胞，迁移效率提高约31.6％；虽然BC006迁移到下室的细胞数目也显著高于作为对照的BC001细胞，迁移效率提高约55.6％，但BC006的迁移能力只有BN003的46.7％。上述结果说明，联合表达CCR2b可以有效介导CAR-T细胞的向CCL2高浓度处迁移。其中，联合表达CCR2b的NKG2D CAR-T细胞的定向迁移能力最高。
出乎意料的是，本发明在Transwell实验中使用的趋化因子浓度和培养时间都与现有技术(实验条件同专利CN111607006B)相当，但检测到的发生迁移的细胞比例(BN003在25ng/ml CCL2诱导6h的条件下，约有71.8％的细胞发生迁移)显著高于现有技术中的数据(该技术的KD-207细胞在25ng/ml CXCL2诱导6h的条件下，最高约有40％的细胞发生迁移)，进一步说明本发明的联合表达CCR2b的NKG2D CAR-T细胞能高效地发生所期望的定向迁移。
实施例5 CAR-T细胞体内抑瘤功能
与CEA CAR-T细胞相比，在NKG2D CAR-T细胞中联合表达CCR2b更能提高趋化迁移的能力。因此，在本实施例中，以BN001和BN003为效应细胞，以SK-OV-3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B-NDG小 鼠进行实验，以观察联合表达CCR2的NKG2D 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B-NDG小鼠进行皮下瘤药效实验，随机分为4组，每组6只，分别为溶媒对照组、Ctrl T对照组、BN001对照组、BN003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2×10 7/ml。在B-NDG小鼠右侧靠近腋下部位皮下注射100μl细胞悬液，即每只小鼠接种2×10 6的靶细胞，接种日记为第0天。
接种靶细胞后第7天(或肿瘤平均体积约为100mm 3时)，通过尾静脉分别注射CAR-T细胞(1×10 7/只)、Ctrl T细胞(1×10 7/只)和溶媒(100μl/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，在第3天、第10天、第28天、第42天采血，加入EDTA抗凝后，用qPCR检测血细胞中CAR-T细胞在小鼠体内的留存情况，通过ELISA检测IFN-γ以监测细胞因子的释放水平。治疗50天后，对小鼠实施安乐死，取肿瘤、心、肝、脾、肺、肾、脑、卵巢等组织称重，每组取2只小鼠的肿瘤组织保存在80℃冰箱，用于提取DNA，检测CAR-T细胞在肿瘤组织中的浸润情况及其在各器官的分布情况；每组取2只小鼠的肿瘤组织进行固定，用HE染色法检测肿瘤细胞的形态和用免疫组化检测组织中抗原的表达情况。
结果如表1所示，在注射CAR-T细胞后50天，溶媒对照组小鼠的平均肿瘤体积约为1588mm 3；Ctrl T对照组小鼠的平均肿瘤体积约为1530mm 3；BN001对照组小鼠的平均肿瘤体积约为409mm 3；BN003实验组小鼠的平均肿瘤体积约为94mm 3。BN003和BN001在体内的抑瘤效果显著优于Ctrl T对照组。同时，BN003的抑瘤效果也进一步显著优于BN001。与BN001相比，注射BN003细胞的小鼠的平均肿瘤体积显著下降，下降幅度约为77％。
用qPCR检测肿瘤组织中的LTR序列，以衡量CAR-T细胞在肿瘤组织中的浸润情况。结果发现，溶媒对照组小鼠的肿瘤组织内LTR的平均本底信号水平为30.5copies/μg DNA；Ctrl T对照组小鼠的肿瘤组织内LTR的平均本底信号水平为25.2copies/μg DNA；BN001对照组小鼠的肿瘤组织内平均LTR含量为11852.0copies/μg DNA；BN003实验组小鼠的肿瘤组织内平均LTR含量为47375.1copies/μg DNA。BN003细胞在肿瘤组织中的浸润程度也显著高于BN001。与BN001相比，BN003细胞向小鼠的皮下移植瘤组织的归巢能力获得显著提升，提升幅度接近300％。以上结果都说明联合表达CCR2的新一代NKG2D CAR-T细胞可高效迁移至肿瘤发生部位，比第二代NKG2D CAR-T细胞在治疗效果上更有优势。
表1 体内药效实验的末次取材检测
Figure PCTCN2022121035-appb-000014
讨论
在针对结直肠癌等实体瘤的CAR-T治疗中，主要识别的靶点有CD133、CEA、EGFR、HER-2和NKG2D配体等。其中，NKG2D(又称CD314)是天然免疫系统中的一个重要的激活性受体，主要表达于自然杀伤细胞、γδT细胞和CD8+T细胞的表面。以NKG2D作为CAR分子的抗原识别结构域具有众多优点。与针对单一靶点的特异性抗体CAR分子不同，基于NKG2D改造获得的CAR分子可以识别至少8种不同的NKG2D配体，包括MICA、MICB、ULBP-1、ULBP-2、ULBP-3、ULBP-4、ULBP-5和ULBP-6，更有利于治疗于异质性高或容易丢失靶点抗原的肿瘤。另外，这些NKG2D配体都位于靶细胞表面。因此，与TCR-T相比，NKG2D CAR-T不需要MHC分子的抗原呈递过程即可直接识别肿瘤细胞。重要的是，NKG2D配体在结直肠癌、卵巢癌、胰腺癌、白血病等上皮源性的肿瘤细胞上有高水平表达，但在正常细胞中不表达或者表达水平极低，是肿瘤特异性治疗的理想靶点。此外，NKG2D CAR-T细胞表面不携带任何外来的可能引发患者免疫反应的蛋白结构，从而降低了CAR-T细胞被患者免疫系统排斥的可能性。
实体瘤细胞可以通过分泌趋化因子CXCL12和CXCL5，阻止CAR-T细胞向肿瘤组织的迁移和浸润。相反，实体瘤细胞较少分泌可促进CAR-T细胞迁移的趋化因子。这两个因素使CAR-T细胞难以到达实体瘤部位。因此，提高CAR-T细胞对肿瘤趋化因子的特异性识别和灵敏度是影响CAR-T治疗效果的关键因素之一，而在CAR-T细胞中联合表达趋化因子受体是解决改问题的重要方法。
趋化因子是一类特殊的细胞因子，包含50多个成员。根据结构分为CC、CXC、CX3C和XC四类；趋化因子受体则相应分为CCR、CXCR、CX3CR和XCR4种，约有20个成员。趋化因子的一种主要作用机制是通过形成可溶性的或固定于基质中的浓度梯度来诱导免疫细胞的定向迁移，从而调免疫细胞在组织中的浸润。目前，有部分CAR-T技术已采用联合表达趋化因子受体的方式来促进CAR-T细胞快速迁移至癌细胞处，从而提高CAR-T细胞的肿瘤治疗效果。
不同类型的实体瘤释放的趋化因子的种类和水平都不尽相同，也有不同的免疫逃逸机制。因此，针对特定癌种选择适合的靶点抗原，再联合表达合适的趋化因子受体，是提高这类型CAR-T细胞治疗效果的关键。
本发明通过特定CAR分子和特定趋化因子受体的特定组合，既高效解决了靶向恶性肿瘤病灶的问题，又有效克服了恶性肿瘤异质性的问题。
一方面，本发明通过CCR2b作为联合表达的趋化因子受体，提高了CAR-T细胞向恶性肿瘤病灶的迁移能力，从而提升了治疗效率。CCR2b主要识别的趋化因子CCL2在结直肠癌、卵巢癌、胰腺癌等多种恶性肿瘤中有异常的高表达，且在正常组织细胞中表达水平极低。同时，CCR2b与CCL2的亲和力极高(约为0.7nM，IC 50数值越小，对应的亲和力和灵敏度越高)，灵敏度远高于其他趋化因子受体和趋化因子的组合(如CCR2a/CCL2组合的IC 50数值约为5nM,CXCR3/CXCL11的组合C 50数值约为8.2nM)。
另一方面，本发明采用NKG2D作为CAR分子的胞外识别结构域，可以识别肿瘤细胞表面的多种靶点抗原(包括MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6)，可降低因肿瘤异质性或抗原丢失而降低疗效的风险，更有利于提高对结直肠癌、卵巢癌、胰腺癌等恶性肿瘤的治疗效果。因此，本发明进一步提升了治疗的有效性，提高了CAR-T细胞等免疫细胞对抗恶性肿瘤的高度异质性的能力。
同时，研究还表明NKG2D CAR-T也会靶向肿瘤微环境中的免疫抑制细胞和新生血管，有助于T细胞等免疫细胞克服免疫抑制的肿瘤微环境，提高肿瘤治疗的效果。
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NKG2D的胞外结构域；和
    (b)所述的免疫细胞表达外源的CCR2b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-NKG2D-H-TM-C-CD3ζ  (I)
    式中，
    L为无或信号肽序列；
    NKG2D为NKG2D胞外结构域或其活性片段；
    H为无或铰链区；
    TM为跨膜结构域；
    C为共刺激信号结构域；
    CD3ζ为源于CD3ζ的胞浆信号传导序列；
    所述“-”连接肽或肽键。
  3. 如权利要求1所述的工程化免疫细胞，其特征在于，所述NKG2D的胞外结构域的氨基酸序列如SEQ ID NO:1第73-216位所示。
  4. 如权利要求1所述的工程化免疫细胞，其特征在于，所述CCR2b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CCR2b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、卵巢癌、胰腺癌。
  9. 如权利要求7所述的用途，其特征在于，所述肿瘤为NKG2D配体高表达和/或趋化因子高表达的肿瘤。
  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NKG2D的胞外结构域；和
    (2)第二核酸序列，所述第二核酸序列含有用于联合表达CCR2b的第二表达盒。
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Publication number Priority date Publication date Assignee Title
CN113621582B (zh) * 2021-09-23 2022-09-02 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
CN113755448B (zh) * 2021-11-10 2022-02-18 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
KR20240099452A (ko) * 2021-11-11 2024-06-28 가부시키가이샤 헤리오스 유전자-변형 다능성 줄기 세포, 이로부터 유래하는 면역담당 세포, 상기 세포의 제조방법 및 이의 용도
CN115212299A (zh) * 2022-06-21 2022-10-21 深圳先进技术研究院 Car-t和car-m联用在制备抗肿瘤药物中的应用
CN117625547B (zh) * 2022-08-25 2024-06-28 广州百吉生物制药有限公司 联合表达gpx4的新一代嵌合抗原受体及其应用
CN117940559A (zh) * 2022-08-25 2024-04-26 广州百吉生物制药有限公司 联合表达cd40l的工程化免疫细胞及其制备和应用

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109153975A (zh) * 2015-12-28 2019-01-04 诺华股份有限公司 制备嵌合抗原受体表达细胞的方法
CN109777784A (zh) * 2019-02-22 2019-05-21 上海尚泰生物技术有限公司 一种增强向肿瘤部位迁移的嵌合抗原受体载体构建方法与应用
CN110872577A (zh) * 2020-01-20 2020-03-10 中国科学院动物研究所 修饰的免疫细胞及其应用
CN111378625A (zh) * 2020-03-18 2020-07-07 华东师范大学 一种cxcl13趋化型car－t细胞的制备和应用
WO2020159941A1 (en) * 2019-01-28 2020-08-06 Xyphos Biosciences Inc. Modified non-natural nkg2d ligands that selectively deliver attached heterologous molecules to non-natural nkg2d receptors on car-cells
CN111632135A (zh) * 2020-05-09 2020-09-08 深圳宾德生物技术有限公司 靶向nkg2d的嵌合抗原受体t细胞在治疗前列腺癌中的应用、治疗前列腺癌的药物
US20200308248A1 (en) * 2019-03-26 2020-10-01 ST Phi Therapeutics Chimeric Natural Killer Cell Receptors and Method of Using Thereof
CN112142854A (zh) * 2020-09-18 2020-12-29 南京凯地生物科技有限公司 免疫调节特异性嵌合抗原受体细胞及制备方法和应用
CN112961248A (zh) * 2021-02-22 2021-06-15 广州百暨基因科技有限公司 共表达IL-7和CCR2b的嵌合抗原受体融合蛋白及其应用
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

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3752197A4 (en) * 2018-04-06 2022-06-08 H. Lee Moffitt Cancer Center & Research Institute, Inc. NKG2D CHIMERIC ANTIGEN RECEPTORS
WO2020190902A1 (en) * 2019-03-19 2020-09-24 H. Lee Moffitt Cancer Center And Research Institute Inc. Chimeric antigen receptors with enhanced tumor infiltration

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109153975A (zh) * 2015-12-28 2019-01-04 诺华股份有限公司 制备嵌合抗原受体表达细胞的方法
WO2020159941A1 (en) * 2019-01-28 2020-08-06 Xyphos Biosciences Inc. Modified non-natural nkg2d ligands that selectively deliver attached heterologous molecules to non-natural nkg2d receptors on car-cells
CN109777784A (zh) * 2019-02-22 2019-05-21 上海尚泰生物技术有限公司 一种增强向肿瘤部位迁移的嵌合抗原受体载体构建方法与应用
US20200308248A1 (en) * 2019-03-26 2020-10-01 ST Phi Therapeutics Chimeric Natural Killer Cell Receptors and Method of Using Thereof
CN110872577A (zh) * 2020-01-20 2020-03-10 中国科学院动物研究所 修饰的免疫细胞及其应用
CN111378625A (zh) * 2020-03-18 2020-07-07 华东师范大学 一种cxcl13趋化型car－t细胞的制备和应用
CN111632135A (zh) * 2020-05-09 2020-09-08 深圳宾德生物技术有限公司 靶向nkg2d的嵌合抗原受体t细胞在治疗前列腺癌中的应用、治疗前列腺癌的药物
CN112142854A (zh) * 2020-09-18 2020-12-29 南京凯地生物科技有限公司 免疫调节特异性嵌合抗原受体细胞及制备方法和应用
CN112961248A (zh) * 2021-02-22 2021-06-15 广州百暨基因科技有限公司 共表达IL-7和CCR2b的嵌合抗原受体融合蛋白及其应用
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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU TAO, LIANG WEIXIANG: "Ultrasound guided NKG2D-CAR gene therapy for advanced rectal cancer： a case report", JOURNAL OF CLINICAL ULTRASOUND IN MEDICINE, vol. 22, no. 9, 30 September 2020 (2020-09-30), XP093054631, ISSN: 1008-6978, DOI: 10.16245/j.cnki.issn1008-6978.2020.09.011 *
WANG YANAN, WANG JING, YANG XINYI, YANG JINLONG, LU PANPAN, ZHAO LIN, LI BOKANG, PAN HANYU, JIANG ZHENGTAO, SHEN XIAOTING, LIANG Z: "Chemokine Receptor CCR2b Enhanced Anti-tumor Function of Chimeric Antigen Receptor T Cells Targeting Mesothelin in a Non-small-cell Lung Carcinoma Model", FRONTIERS IN IMMUNOLOGY, vol. 12, 1 January 2021 (2021-01-01), pages 628906, XP093054444, DOI: 10.3389/fimmu.2021.628906 *

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