WO2024037288A1 - 一种靶向人dll3抗原的第四代car及其载体的制备和应用 - Google Patents

一种靶向人dll3抗原的第四代car及其载体的制备和应用 Download PDF

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WO2024037288A1
WO2024037288A1 PCT/CN2023/108807 CN2023108807W WO2024037288A1 WO 2024037288 A1 WO2024037288 A1 WO 2024037288A1 CN 2023108807 W CN2023108807 W CN 2023108807W WO 2024037288 A1 WO2024037288 A1 WO 2024037288A1
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dll3
cells
car
antigen
amino acid
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PCT/CN2023/108807
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English (en)
French (fr)
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陈皓
文献
王晚秋
董创创
窦昌林
仉慧敏
冯健霞
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南京博安生物技术有限公司
山东博安生物技术股份有限公司
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Publication of WO2024037288A1 publication Critical patent/WO2024037288A1/zh

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    • 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
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to the field of biomedicine or biopharmaceutical technology, and in particular to a CAR structure targeting human DLL3 antigen that co-expresses immunosuppressive molecules and cytokines, its preparation method and its use in the preparation of cell drugs (such as T cells, NK cells). , macrophages and other human cells with immune functions), treatment and prevention of diseases.
  • a CAR structure targeting human DLL3 antigen that co-expresses immunosuppressive molecules and cytokines its preparation method and its use in the preparation of cell drugs (such as T cells, NK cells). , macrophages and other human cells with immune functions), treatment and prevention of diseases.
  • Chimeric antigen receptor Chimeric antigen receptor, CAR
  • CAR-T cell therapy technology is an adoptive immune cell technology that has developed very rapidly in recent years. Through genetic modification technology, the targeting, killing activity and persistence of effector T cells are superior to conventionally used immune cells, and they can overcome the local immunosuppressive microenvironment of the tumor and break the host immune tolerance state. It is an ideal tumor immune cell therapy. New targeted treatments in the field.
  • Human DLL3 protein (Delta-Like Ligand 3, DLL3) is a single-pass transmembrane protein composed of 619 amino acids and belongs to the Notch ligand family. It is a highly tumor-selective cell surface target that is highly expressed in most small cell lung cancer (SCLC) and carcinoid subgroups (Lung Cancer; 135:73-79; 2019), but is not expressed in normal lung cancer tissues and cancer cells. Not expressed in paratissues. In one study, independent tumor specimens from 1,073 SLCL patients showed that 85% had positive DLL3 expression ( ⁇ 25%) and 68% had high DLL3 expression ( ⁇ 75%) (Lung Cancer; 147:237-243 ;2020). Chimeric antigen receptor T cells targeting human DLL3 can stimulate T cells to specifically kill tumor cells by recognizing the DLL3 antigen on the tumor surface.
  • CAR-T solid tumor treatment mainly has the following major problems: 1. Lack of effective targets; 2. CAR-T transport and Infiltration; 3. Immunosuppression of the tumor microenvironment; 4. Endogenous T cell inhibitory signals.
  • the present invention gives immune cells more functions by optimizing the CAR vector structure, especially the CAR structure targeting the human DLL3 antigen that co-expresses immunosuppressive molecules and /h or factors, and is expected to have a therapeutic effect on patients with DLL3 antigen-positive tumors. Come to hope.
  • drugs targeting cancers that highly express DLL3, such as the treatment of lung cancer, melanoma, medullary thyroid cancer, glioblastoma, prostate cancer, and neuroendocrine cancer.
  • the present invention provides a chimeric antigen receptor (CAR) targeting DLL3, and immune cells containing the CAR, including but Not limited to T cells.
  • CAR chimeric antigen receptor
  • the invention also provides nucleic acid encoding the CAR; expression cassettes, vectors, and cells containing the nucleic acid; pharmaceutical compositions containing the CAR, the nucleic acid, the expression cassette, the vector, and the cells; A kit containing the CAR, the nucleic acid, the expression cassette, the vector, the cell, and the pharmaceutical composition; the CAR, the immune cell containing the CAR, the nucleic acid, and the expression
  • the application of the box, the vector, the cells, and the pharmaceutical composition in preventing, treating, detecting or diagnosing diseases related to DLL3, or in preparing drugs or preparations for preventing and/or treating cancer or tumors, so
  • the disease related to DLL3 is a disease with high expression of DLL3, further the disease is a cancer or tumor with high expression of DLL3, and further, the cancer or tumor is selected from the group consisting of lung cancer, melanoma, medullary thyroid cancer, glioblastoma, One or more of prostate cancer and neuroendocrine cancer; further
  • a first aspect of the present invention provides an anti-DLL3 antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising the following three light chain complementarity-determining regions and/or three heavy chain complementarity-determining regions, and the antibody Or the three light chain complementarity determining regions of its antigen-binding fragment include LCDR1 as shown in SEQ ID NO:22, LCDR2 as shown in SEQ ID NO:23, LCDR3 as shown in SEQ ID NO:24; and/or 3 heavy
  • the chain complementarity determining region includes HCDR1 as shown in SEQ ID NO:18, HCDR2 as shown in SEQ ID NO:19, and HCDR3 as shown in SEQ ID NO:20.
  • the antibody or its antigen-binding fragment is characterized in that the antibody or its antigen-binding fragment contains less than 95%, 96%, 97%, 98%, or less than the amino acid sequence shown in SEQ ID NO:21.
  • a light chain variable region (VL) that is 99% or 100% identical, and has at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence set forth in SEQ ID NO:17 Heavy chain variable region (VH).
  • the antibody includes a heavy chain constant region that is less than 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in SEQ ID NO: 25, and includes a heavy chain constant region that is identical to SEQ ID NO: 25.
  • the sequence shown in 26 has less than 95%, 96%, 97%, 98%, 99% or 100% identity to the light chain constant region.
  • a second aspect of the present invention a chimeric antigen receptor (CAR) targeting DLL3 antigen, the CAR comprising a DLL3 antigen binding domain, a transmembrane domain and an intracellular signal transduction domain, wherein the DLL3 antigen
  • the binding domain is a scFv, wherein the scFv is the antibody or antigen-binding fragment thereof according to any one of claims 1-2.
  • the chimeric antigen receptor targeting DLL3 antigen is characterized in that the CAR also includes one or more of a hinge region, a signal peptide and a costimulatory signal domain;
  • the transmembrane domain is a CD8 transmembrane region
  • the hinge region is a CD8 hinge region
  • the intracellular signaling domain is a CD3 ⁇ intracellular signaling domain
  • the signal peptide is a CD8 ⁇ signal peptide
  • the The costimulatory signal domain is 4-1BB or CD28 costimulatory signal domain.
  • the chimeric antigen receptor targeting DLL3 antigen is characterized in that the CAR sequentially includes CD8 ⁇ signal peptide, DLL3 antibody scFv VH-linker-DLL3 antibody scFv VL, and CD8 hinge region from N-terminus to C-terminus. , CD8 transmembrane region, 4-1BB costimulatory signal and CD3 ⁇ intracellular signaling domain.
  • chimeric antigen receptor targeting DLL3 antigen is characterized by:
  • the CD8 ⁇ signal peptide includes an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 27;
  • the CD8 hinge region and transmembrane region comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 28;
  • the 4-1BB costimulatory signal domain includes an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 29; or
  • the CD3 ⁇ intracellular signaling domain includes an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 30;
  • amino acid sequence of the CAR is shown in SEQ ID NO: 32.
  • the chimeric antigen receptor targeting DLL3 antigen is characterized in that the CAR includes the following elements i) and/or ii):
  • the immunosuppressive molecule is selected from one or more of PD1 and PDL1;
  • the cell membrane-type interleukin is selected from the group consisting of cell membrane-type IL2 cytokines, cell membrane-type IL4 cytokines, cell membrane-type IL7 cytokines, and cell membrane-type IL9 cells.
  • the secreted chemokine was selected from secreted CCL1 chemokine, secreted CCL2 chemokine, secreted CCL3 chemokine, secreted CCL5 chemokine, secreted CCL7 chemokine, secreted CCL15 chemokine, secreted CCL16 chemokine.
  • Chemokine secreted CCL19 chemokine, secreted CCL20 chemokine, secreted CCL21 chemokine, secreted CXCL4 chemokine, secreted CXCL9 chemokine, secreted CXCL10 chemokine, secreted CXCL11 chemokine
  • the chimeric antigen receptor targeting DLL3 antigen includes anti-PDL1 protein expression elements and/or cell membrane IL7 cytokine expression elements;
  • the anti-PDL1 expression element contains kappa leader signal peptide, anti-PD-L1 antibody scfv, linker peptide 1 and human IgG CH2CH3 fragment in sequence;
  • the cell membrane type IL7 cytokine expression element contains human IL-7 cytokine fragment in sequence. , connecting peptide 2, CD8 transmembrane region;
  • the kappa leading signal peptide in the anti-PDL1 expression element includes an amino acid with at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO:33.
  • Sequence; anti-PDL1 antibody scfv contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO:34;
  • the amino acid sequence of connecting peptide 1 is GGGS ;
  • the human IgG CH2CH3 fragment includes an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 35; in the cell membrane type IL7 cytokine expression element
  • the human IL-7 cytokine fragment includes an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 36;
  • the chimeric antigen receptor targeting DLL3 antigen contains an anti-PDL1 protein expression element. Further, the amino acid sequence of the CAR is shown in SEQ ID NO: 43.
  • the chimeric antigen receptor targeting DLL3 antigen contains a cell membrane-type IL7 cytokine expression element, and further, the amino acid sequence of the CAR is shown in SEQ ID NO: 44.
  • the chimeric antigen receptor targeting DLL3 antigen includes an anti-PDL1 protein expression element and a cell membrane IL7 cytokine expression element. Furthermore, the amino acid sequence of the CAR is as SEQ ID NO:45 shown.
  • a third aspect of the present invention provides a nucleic acid encoding the anti-DLL3 antibody or antigen-binding fragment thereof or encoding the chimeric antigen receptor targeting the DLL3 antigen.
  • a fourth aspect of the present invention provides an expression cassette comprising the nucleic acid.
  • a fifth aspect of the present invention provides a vector containing the nucleic acid or the expression cassette.
  • a sixth aspect of the present invention provides a nucleic acid encoding the anti-DLL3 antibody or antigen-binding fragment thereof or CAR.
  • a sixth aspect of the invention provides an expression cassette comprising said nucleic acid.
  • the seventh aspect of the present invention provides a vector comprising a nucleic acid encoding the anti-DLL3 antibody or an antigen-binding fragment thereof, a nucleic acid of a CAR, or the expression cassette.
  • the vector can be used to express the anti-DLL3 antibody or antigen-binding fragment thereof or to express the CAR.
  • the vector can be a viral vector;
  • the viral vector includes but is not limited to lentiviral vector, adenoviral vector, adeno-associated virus vector or retroviral vector, etc.;
  • the vector can be non-viral Vector; preferably, the vector can be a mammalian cell expression vector; preferably, the expression vector can be a bacterial expression vector; preferably, the expression vector can be a fungal expression vector.
  • the eighth aspect of the present invention provides a cell, the cell comprising the nucleic acid, or the expression cassette, or the vector, and the cell can express the anti-DLL3 antibody or antigen-binding fragment thereof or the CAR .
  • the cells are bacterial cells;
  • the bacterial cells are E. coli cells, etc.;
  • the cells are fungal cells;
  • the fungal cells are yeast cells;
  • the yeast The cells are Pichia pastoris cells, etc.;
  • the cells are mammalian cells; preferably, the mammalian cells are Chinese hamster ovary cells (CHO), human embryonic kidney cells (293), B cells, T cells, DC cells or NK cells, etc.
  • the cells are Engineered immune cells; more preferably, the engineered immune cells are T cells; most preferably, the T cells are primary source T cells or T cells differentiated from iPSCs, and the T cells differentiated from IPSCs
  • the cells are ⁇ T cells, DNT cells or NKT cells.
  • the ninth aspect of the present invention provides a pharmaceutical composition comprising the CAR, nucleic acid, expression cassette, vector or cell.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes one or more of the following: pharmaceutically acceptable solvents, dispersants, additives, shaping agents, and pharmaceutical excipients.
  • the tenth aspect of the present invention provides a kit comprising the anti-DLL3 antibody of the present invention or its antigen-binding fragment, the CAR, or a nucleic acid encoding a CAR, or the expression cassette.
  • the eleventh aspect of the present invention provides the use of the anti-DLL3 antibody or antigen-binding fragment thereof, the CAR, nucleic acid, expression cassette, vector or cell in the preparation of a pharmaceutical composition for treating or preventing diseases.
  • the twelfth aspect of the present invention provides the use of the anti-DLL3 antibody or antigen-binding fragment thereof, the CAR or nucleic acid or expression cassette in the preparation of diagnostic and detection kits.
  • the thirteenth aspect of the present invention provides a method for treating or preventing diseases, comprising combining the anti-DLL3 antibody or antigen-binding fragment thereof, the CAR, nucleic acid, expression cassette, vector, cell or drug of the present invention Materials are given to subjects in need.
  • the fourteenth aspect of the present invention provides a method for diagnosis and detection, including administering the anti-DLL3 antibody or antigen-binding fragment thereof, the CAR, nucleic acid, expression cassette, kit or pharmaceutical composition of the present invention Subjects or samples in need.
  • the fifteenth aspect of the present invention provides the use of the anti-DLL3 antibody or antigen-binding fragment thereof, the CAR, nucleic acid, expression cassette, vector, cell or pharmaceutical composition for treating and preventing diseases.
  • the sixteenth aspect of the present invention provides the use of the anti-DLL3 antibody or antigen-binding fragment thereof, the CAR, nucleic acid, expression cassette, kit, or pharmaceutical composition for detection and diagnosis.
  • the seventeenth aspect of the present invention provides the anti-DLL3 antibody or antigen-binding fragment thereof, the CAR, the nucleic acid, or the expression cassette, or the vector, or the pharmaceutical composition for prevention, Applications to treat, detect or diagnose DLL3-related diseases.
  • the disease related to DLL3 is a disease with high expression of DLL3; preferably, the disease is a cancer or tumor with high expression of DLL3; more preferably, the cancer or tumor is selected from lung cancer, melanoma, One or more of medullary thyroid cancer, glioblastoma, prostate cancer, and neuroendocrine cancer; most preferably, the cancer is lung cancer, especially small cell lung cancer.
  • the eighteenth aspect of the present invention provides a method for preparing engineered immune cells, which is characterized by comprising the following steps:
  • the immune cells are T cells; more preferably, the T cells are primary T cells or T cells differentiated from iPSCs, and the T cells differentiated from iPSCs are ⁇ T cells, DNT cells or NKT cells. .
  • a nineteenth aspect of the present invention also provides the use of DLL3 antibodies or antigen-binding fragments thereof in the preparation of antibody-drug conjugates (ADCs).
  • ADCs antibody-drug conjugates
  • a twentieth aspect of the present invention provides an anti-DLL3 antibody-drug conjugate, which includes (a) the anti-DLL3 antibody or antigen-binding fragment thereof of the present invention, and (b) and The antibody moiety is coupled to a coupling moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.
  • the drug is selected from the following group: chemotherapy drugs, radiotherapy drugs, hormone therapy drugs or immunotherapy drugs.
  • the fourth generation CAR targeting human DLL3 antigen and its vector provided by the present invention especially the CAR vector targeting human DLL3 antigen that co-expresses anti-PD1 or PD-L1 fusion protein and/or interleukin fusion protein, has the following characteristics:
  • the anti-DLL3 antibody or antigen-binding fragment thereof provided by the present invention has good affinity to both human DLL3 protein and DLL3-expressing cells.
  • the recombinant plasmid expressing DLL3 CAR constructed by the present invention, the recombinant plasmid expressing DLL3 CAR and anti-PD-L1 fusion protein (DLL3 CAR.aPDL1), and the recombinant plasmid expressing DLL3 CAR and cell membrane IL7 fusion protein ( DLL3 CAR.mIL7) and a recombinant plasmid that co-expresses DLL3 CAR, anti-PD-L1 scfv fusion protein and cell membrane IL7 fusion protein (DLL3 All four plasmids (CAR.aPDL1.mIL7) can be used to prepare CAR-T cells, and the CAR positivity rate ranges from 10% to 60%.
  • DLL3 CAR-T cells DLL3 CAR.aPDL1-T cells
  • DLL3 CAR-T cells DLL3 CAR-T cells
  • CAR.mIL7-T cells DLL3 CAR-T cells that stably co-express anti-PD-L1 and IL7 fusion proteins
  • the CAR-T cells co-expressing mIL7 fusion protein constructed in the present invention can enhance the vitality and persistence of CAR-T cells.
  • the mIL7 fusion protein mainly acts on the T cell's own CAR-positive cell population and triggers the expansion of its own CAR-positive cell population. and remain active.
  • DLL3 CAR-T cells expressing different functional structures can specifically kill tumor cells expressing human DLL3 antigen, and co-expression of aPDL1 fusion protein and mIL7 protein does not affect the killing effect of DLL3 CAR-T cells.
  • the cytokine IFN- ⁇ release level is at least 2 times higher than that of the negative control (T cells without CAR transduction), and compared with DLL3 CAR-T, co-expression of aPDL1 fusion protein and mIL7 protein did not affect IFN- ⁇ factor release from DLL3 CAR-T cells.
  • the DLL3 CAR.mIL7-T cells and DLL3 CAR.aPDL1.mIL7-T cells constructed in the present invention that co-express the mIL7 fusion protein have a significantly better clearing effect on tumor cells than the DLL3 CAR-T cells that do not co-express the IL7 fusion protein.
  • DLL3 CAR.aPDL1-T cells; and DLL3 CAR.mIL7-T cells and DLL3 CAR.aPDL1.mIL7-T cells that co-expressed mIL7 fusion protein maintained good cell expansion during sustained killing, and their cell persistence was excellent.
  • DLL3 CAR-T and DLL3 CAR.aPDL1-T cells that do not co-express IL7 fusion protein In DLL3 CAR-T and DLL3 CAR.aPDL1-T cells that do not co-express IL7 fusion protein.
  • the aPDL1 fusion protein secreted by DLL3 CAR-T cells co-expressing aPDL1 fusion protein in the present invention can bind to the PD-L1 protein of tumor cells, thereby blocking the PD-1 and PD-L1 pathways.
  • DLL3 CAR-T cells co-expressing mIL7 fusion protein can enhance the expansion ability and persistence of CAR-T cells.
  • DLL3 CAR-T cells that co-express aPDL1 fusion protein and mIL7 fusion protein retain the functions of the above two structures and enhance the ability of DLL3 CAR-T cells to continuously kill tumors.
  • DLL3 CAR-T cells and DLL3 CAR.aPDL1.mIL7-T cells both had inhibitory effects on SHP-77 tumors in mice; co-expression DLL3 CAR-T cells with aPDL1 fusion protein and mIL7 fusion protein showed better tumor suppression effect at low doses, indicating that co-expression of aPDL1 fusion protein and mIL7 fusion protein can significantly enhance the anti-tumor ability of DLL3 CAR-T cells.
  • the present invention constructs a fourth-generation CAR and its vector to co-express anti-PD-L1 scfv fusion protein (aPDL1) and/or and cell membrane IL7 fusion protein (mIL7).
  • aPDL1 scfv fusion protein can block the PD-1/PD-L1 signaling pathway, reduce T cell exhaustion, and maintain T cell stability.
  • the cell membrane IL7 fusion protein enhances T Cell viability and expansion ability, thereby solving the problems of CAR-T cell immunosuppression in the tumor microenvironment and poor persistence in the body.
  • Figure 1 shows the serum dilution ratios of 500x, 2500x, 12500x, and 62500x in the serum titers of mice immunized with DLL3 protein in Example 1.
  • Figure 2 shows the ELISA binding activity results of the antibody of Example 1 and human DLL3-His protein.
  • Figure 3 is the results of the binding activity of the antibody to CT26-hDLL3 cells in Example 1.
  • Figure 4A-4C is a schematic diagram of the gene fragment and vector structure in Example 2, wherein Figure 4A is the structure of the DLL3 CAR, DLL3 CAR.aPDL1, DLL3 CAR mIL7, DLL3 CAR.aPDL1.mIL7 gene fragment; Figure 4B is the recombinant plasmid pRRLSIN-DLL3 Schematic diagram of the structure of CAR (PB DLL3 CAR); Figure 4C is a schematic structural diagram of the recombinant plasmid pRRLSIN-DLL3 CAR.aPDL1.mIL7 (PB DLL3 CAR.aPDL1.mIL7).
  • Figure 4A is the structure of the DLL3 CAR, DLL3 CAR.aPDL1, DLL3 CAR mIL7, DLL3 CAR.aPDL1.mIL7 gene fragment
  • Figure 4B is the recombinant plasmid pRRLSIN-DLL3 Schematic diagram of the structure of CAR (PB
  • Figure 5A- Figure 5E are schematic flow cytometry diagrams of CAR positive detection of CAR-T cells expressing different functional structures of DLL3 by anti-human IgG (Fab) 2 antibodies in Example 3, wherein Figure 5A shows the results of T cells without CAR transduction , Figure 5B shows the results for DLL3 CAR-T cells, Figure 5C shows the results for DLL3 CAR.aPDL1-T cells, Figure 5D shows the results for DLL3 CAR.mIL7-T cells, and Figure 5E shows the results for DLL3 Results for CAR.aPDL1.mIL7-T cells.
  • Fab anti-human IgG
  • Figure 6A- Figure 6E are CAR positive flow cytometry diagrams of CAR-T cells expressing different functional structures of DLL3 detected by DLL3 antigen in Example 3, wherein Figure 6A shows the results of T cells without CAR transduction, and Figure 6B shows Results for DLL3 CAR-T cells, Figure 6C shows the results for DLL3 CAR.aPDL1-T cells, Figure 6D shows the results for DLL3 CAR.mIL7-T cells, Figure 6E shows the results for DLL3 CAR.aPDL1.mIL7- T cell results.
  • Figure 7A shows the detection of CAR positivity of DLL3 CAR-T cells with different functional structures by the anti-human IgG (Fab) 2 antibody detection method in Example 3. Rate results; Figure 7B shows the CAR positivity rate results of DLL3 CAR-T cells with different functional structures detected by the human DLL3 antigen protein detection method in Example 3.
  • Fab anti-human IgG
  • Figure 8A shows the change results of the CAR positive rate of DLL3 CAR-T cells with different functional structures under different culture days using the anti-human IgG (Fab) 2 antibody detection method in Example 3;
  • Figure 8B shows the detection of human DLL3 antigen protein in Example 3 Methods The changes in CAR positive rate of DLL3 CAR-T cells with different functional structures under different culture days were detected.
  • Fab anti-human IgG
  • Figure 9A- Figure 9E are flow cytometry diagrams of detection of IL7 protein expression on the cell membrane surface of T lymphocytes expressing different structures of DLL3 CAR by anti-human IL7 antibodies in Example 4, wherein Figure 9A shows the results of T cells without CAR transduction , Figure 9B shows the results for DLL3 CAR-T cells, Figure 9C shows the results for DLL3 CAR.aPDL1-T cells, Figure 9D shows the results for DLL3 CAR.mIL7-T cells, and Figure 9E shows the results for DLL3 Results for CAR.aPDL1.mIL7-T cells.
  • Figures 10A to 10J are flow cytometry diagrams showing the binding reaction of CAR-T cell culture supernatants expressing different functional structures of DLL3 to different tumor cells in Example 5.
  • Figures 10A-10E show a flow cytometry diagram of the binding reaction of CAR-T cell culture supernatants expressing different functional structures of DLL3 to SHP-77-hDLL3 cells
  • Figures 10F-10J show CAR-T cells expressing different functional structures of DLL3 Flow cytometry diagram of the binding reaction of culture supernatant to SHP-77-hDLL3-hPDL1 cells.
  • Figure 11 shows the test results of the concentration of human IgG protein (aPDL1) in the culture supernatant of DLL3 CAR-T cells expressing different functional structures in Example 5.
  • Figure 12 shows the results of cell viability changes in different days of culture of DLL3 CAR-T cells expressing different functional structures in factor-free conditions in Example 6.
  • Figure 13 shows the results of changes in the number of viable cells in different days of culture of DLL3 CAR-T cells expressing different functional structures in factor-free conditions in Example 6.
  • Figure 14 shows the results of changes in the CAR positive rate of T cells expressing DLL3 CARs with different functional structures in culture in factor-free conditions for different days in Example 6.
  • Figure 15A- Figure 15D are the in vitro specific killing results of lung cancer cell lines by DLL3 CAR-T cells expressing different functional structures in Example 7.
  • Figure 15A shows the effect of DLL3 CAR-T cells expressing different functional structures on NCI-H460 In vitro specific killing results of cell lines.
  • Figure 15B shows the in vitro specific killing results of SHP-77 cell lines by DLL3 CAR-T cells expressing different functional structures.
  • Figure 15C shows the DLL3 CAR-T cells expressing different functional structures. In vitro specific killing results of T cells against human DLL3-positive SHP-77-hDLL3 cell line.
  • FIG 15D shows the DLL3 CAR-T cells expressing different functional structures against human DLL3-positive and human PD-L1-positive SHP-77- In vitro specific killing results of hDLL3-hPDL1 cell line, T in Figure 15A- Figure 15D represents the experimental results of T cells without CAR transduction.
  • Figure 16A- Figure 16D show the results of IFN- ⁇ release after DLL3 CAR-T cells expressing different functional structures killed four lung cancer cells in Implementation 7, wherein Figure 16A shows DLL3 CAR-T expressing different functional structures and NCI- IFN- ⁇ release after co-culture of H460 cell line.
  • Figure 16B shows the IFN- ⁇ release after co-culture of DLL3 CAR-T expressing different functional structures and SHP-77 cell line.
  • Figure 16C shows the IFN- ⁇ release after co-culture of DLL3 CAR-T expressing different functional structures. IFN- ⁇ release after co-culture of DLL3 CAR-T and SHP-77-hDLL3 cell lines.
  • Figure 16D shows the release of IFN- ⁇ after co-culture of DLL3 CAR-T expressing different functional structures and SHP-77-hDLL3-hPDL1 cell lines.
  • IFN- ⁇ release, T in Figures 16A-16D represents IFN- ⁇ release from T cells without CAR transduction.
  • Figure 17A- Figure 17B are the results of the long-term anti-tumor effect of DLL3 CAR-T expressing different functional structures on DLL3-expressing positive cells SHP-77 in Example 8, wherein Figure 17A shows the effect of DLL3 CAR-T cells expressing different functional structures on DLL3-positive cells SHP-77 The number of tumor cells changes in the long-term anti-tumor effect of SHP-77 on DLL3-expressing cells.
  • Figure 17B shows the number of CAR-positive T cells expressing the long-term anti-tumor effect of DLL3 CAR-T expressing different functional structures on DLL3-expressing cells. Change results.
  • Figure 18 shows the PD-L1 protein expression results of SHP-77 cells in Example 8 after sustained killing by DLL3 CAR-T cells expressing different functional structures.
  • Figure 19 shows the results of tumor size changes in small cell lung cancer SHP-77 tumor-bearing NCG mice after administration of DLL3 CAR-T cells, DLL3 CAR.aPDL1.mIL7-T cells and non-CAR-transduced T cells in Example 9 .
  • DLL3 antigen proteins with two tags, DLL3-Fc and DLL3-His were immunized into mice by abdominal subcutaneous injection. Freund's complete adjuvant was used to emulsify the antigen for the first immunization, and Freund's incomplete adjuvant was used for the second and fourth immunizations. emulsified antigen.
  • Figure 1 shows the serum titers of mice immunized with DLL3 protein.
  • 500x, 2500x, 12500x, and 62500x represent serum dilution ratios.
  • mice were sacrificed, and the spleens were removed by dissection.
  • the spleens were ground and broken with a syringe stopper and filtered with a filter.
  • the filtered splenocytes were frozen and prepared.
  • cDNA was obtained.
  • the establishment of the phage library was carried out according to conventional methods.
  • the storage capacity data of the built library is shown in Table 1-2.
  • the antibody variable region genes were amplified by conventional molecular biology technology PCR (2 ⁇ Phanta Max Master Mix manufacturer: Vazyme Catalog number: P515-P1-AA batch number: 7E351H9), and the antibody heavy chain variable region genes were separately separated through homologous recombination.
  • the vector pCDNA3.4 (Life Technology) containing the nucleic acid sequence of the antibody heavy chain constant region sequence was ligated into the vector pCDNA3.4 (Life Technology), and the antibody light chain variable region gene was ligated into the vector pCDNA3.4 containing the nucleic acid sequence of the antibody light chain constant region sequence.
  • the variable region sequences of each antibody used to construct positive clone IgG1 in the examples of this application are shown in Tables 1-4. Chain and light chain constant region sequences are shown in Tables 1-5.
  • sequenced positive clones were extracted with plasmids and co-transfected into HEK293 cells, cultured in a shaker at 37°C ⁇ 8% CO2 ⁇ 125rpm. After transient expression for 7 days, the supernatant was passed through Protein A affinity chromatography, and the antibody was purified and passed through UV280 combined with theoretical extinction coefficient determines antibody concentration.
  • VL and VH are CDR, and the analysis system is the IMGT system.
  • Figure 2 shows the ELISA binding activity of the antibody to human DLL3-His protein. The results It was shown that the three antibodies with antibody IDs 229, 244 and 359 all showed better binding activity to human DLL3-His protein.
  • FIG. 3 shows the binding activity of the antibody to CT26-hDLL3 cells.
  • the Isotype in Figure 3 is a self-made irrelevant IgG1 antibody of other targets, which is used as a blank control. Isotype does not show binding activity. Taking the antibody concentration as 10 ⁇ g/mL as an example, the MFI of Isotype is 10120, the MFI of the antibody group is 26276-78408, and the MFI of the antibody group is 2.6-7.7 times that of Isotype.
  • the EC 50 value is shown in Table 1-6 .
  • the antibody DL3-BA359 specifically binds to CT26-hDLL3 cells and has high binding activity.
  • the binding EC 50 is 0.066, which is better than the antibodies with antibody numbers DL3-BA244 and DL3-BA229.
  • Table 1-6 EC 50 of each antibody binding to CT26-hDLL3 cells
  • Human DLL3-His protein was diluted with 1x HBS-EP + Buffer 2-fold gradient to 5 concentrations, namely 50nM, 25nM, 12.5nM, 6.25nM and 3.125nM. Dilute the antibody sample to 2 ⁇ g/mL with 1x HBS-EP + , capture the antibody sample with ProA chip, set the instrument capture time to 70s, flow rate 10 ⁇ L/min; analyte: binding 60s, flow rate 30 ⁇ L/min, dissociation 600s; regeneration: Regenerate with 10mM Glycine-HCl buffer at pH 1.5 for 30s, flow rate 30 ⁇ L/min, Startup 3 times.
  • the equilibrium dissociation constant (KD) was calculated using the 1:1 binding model (BIAcore Insight Evaluation Software version 2.0.15.12933).
  • the affinity results of each antibody are shown in Table 1-7.
  • the KD of all antibodies is less than 1.0.
  • E-09 shows higher affinity, among which the antibody numbered BA359 Demonstrate the highest level of affinity.
  • the present invention designs the targeted DLL3 chimeric antigen receptor gene fragment in the order of the following coding genes: CD8 ⁇ signal peptide, DLL3 antibody scFv VH-linker-DLL3 antibody scFv VL, CD8 hinge region, CD8 transmembrane region and 4-1BB costimulation signal and CD3 ⁇ intracellular signaling domain.
  • CD8 ⁇ signal peptide DLL3 antibody scFv VH-linker-DLL3 antibody scFv VL
  • CD8 hinge region CD8 transmembrane region
  • 4-1BB costimulation signal CD3 ⁇ intracellular signaling domain
  • the DLL3-targeted chimeric antigen receptor gene fragment was prepared through gene synthesis technology. Its amino acid sequence is shown in SEQ ID NO: 32. The specific sequence is shown in Table 2-2.
  • the aPDL1 fusion protein gene fragment contains the following genes: kappa leader signal peptide, anti-PD-L1 antibody scfv, linker peptide 1, and human IgG CH2CH3 fragment.
  • the amino acid sequence of connecting peptide 1 is G4S, which is GGGGS
  • the amino acid sequence of kappa leader signal peptide is SEQ ID NO:33
  • the amino acid sequence of anti-PD-L1 antibody scfv is SEQ ID NO:34
  • the amino acid sequence of human IgG CH2CH3 fragment is SEQ ID NO:35.
  • the mIL7 fusion protein gene fragment contains the following genes: human IL-7 cytokine fragment, linker peptide 2, and CD8 transmembrane region.
  • the amino acid sequence of the linking peptide 2 is Ser-Gly Linker, namely SGGGSGGGGSGGGGSGGGGSGGGSLQ, the amino acid sequence of the human IL-7 cytokine fragment is SEQ ID NO:36, and the amino acid sequence of the CD8 transmembrane region is SEQ ID NO:37.
  • the aPDL1-mIL7 fusion protein gene fragment contains aPDL1 and mIL7 fusion protein gene fragments, which are connected by a 2A peptide.
  • the 2A peptide is (GSG)T2A, and the amino acid sequence is (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO: 38).
  • the specific sequence of SEQ ID NO:33-37 is shown in Table 2-3.
  • aPDL1, mIL7, and aPDL1-mIL7 fusion protein genes were synthesized by Nanjing GenScript Biotechnology Co., Ltd. through gene synthesis technology. Their amino acid sequences are shown in SEQ ID NO: 21-23. The specific sequences are shown in Table 2-4.
  • the anti-PD-L1 fusion protein (aPDL1) sequence or cell membrane IL7 fusion protein (mIL7) sequence or aPDL1-mIL7 fusion protein sequence prepared in Part 2.2 above was inserted into DLL3 through homologous recombination on the Piggybac DLL3 CAR plasmid structure. After the CAR sequence, a 2A peptide is connected.
  • the 2A peptide is (GSG)P2A, and the amino acid sequence is (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO: 42), forming DLL3 CAR.aPDL1, DLL3 CAR.mIL7, and DLL3 CAR.aPDL1.mIL7
  • the gene fragment structure, its amino acid sequence is SEQ ID NO:43-45, the specific sequence is shown in Table 2-5, and the structural diagram is shown in Figure 4A.
  • the vectors constructed above form three plasmid vectors: Piggybac DLL3 CAR-aPDL1 plasmid vector, Piggybac DLL3 CAR-mIL7 plasmid vector, Piggybac DLL3 CAR-aPDL1-mIL7 plasmid vector (respectively referred to as DLL3 CAR.aPDL1, DLL3 CAR.mIL7, DLL3 CAR.aPDL1.mIL7).
  • Figure 4B shows a schematic diagram of the DLL3 CAR plasmid structure under the piggybac (PB) transposon system
  • Figure 4C shows a schematic diagram of the DLL3 CAR.aPDL1.mIL7 plasmid structure under the piggybac (PB) transposon system.
  • PBMC Peripheral blood mononuclear cells
  • CD3MicroBeads human-lyophilized Kit Miltenyi Biotech
  • high-purity CD3-positive T lymphocytes were positively sorted.
  • the sorted CD3 The proportion of positive T cells is above 95%.
  • the purified T cells were then used to activate and proliferate T lymphocytes using Dynabeads Human T-Activator CD3/CD28 (purchased from Thermo Fisher, Cat. No. 111.32D).
  • Transposons also known as transposable elements, are a type of genetic element that can "jump and move" within the genome.
  • PiggyBac transposon (PB transposon for short) has a wide range of transposition activities and can achieve efficient gene transposition with less reliance on host factors.
  • Gene transduction based on the piggybac (PB) system consists of two parts: PB transposase (in DNA or mRNA form) and a plasmid carrying the target gene (PB plasmid).
  • PB transposase in DNA or mRNA form
  • PB plasmid plasmid carrying the target gene
  • the PB system does not use viruses and can deliver CAR-expressing transgenes into T cells, and can carry more genetic material without the risks and safety associated with lentiviral transduction systems. higher.
  • the PB plasmids obtained in 2.3 DLL3 CAR, DLL3 CAR.aPDL1, DLL3 CAR.mIL7, DLL3 CAR.aPDL1.mIL7PB plasmid were used for electroporation. Methods as below:
  • DLL3 CAR DLL3 CAR.aPDL1, DLL3 CAR.mIL7, and DLL3 CAR.aPDL1.mIL7 plasmids were obtained (the above cells are referred to as: DLL3 CAR-T, DLL3 CAR.aPDL1 -T, DLL3 CAR.mIL7-T, DLL3 CAR.aPDL1.mIL7-T) and T cell controls without CAR plasmid electroporation were used for flow cytometric analysis of CAR positivity rate.
  • the analysis method is as follows:
  • the experimental results show that the DLL3 CAR, DLL3 CAR.aPDL1, DLL3 CAR.mIL7, and DLL3 CAR.aPDL1.mIL7 structures in the present invention can be used to prepare CAR-T cells, and the DLL3 CAR-T co-expressing aPDL1 and mIL7 fusion protein structures
  • the CAR positive rate of cells was comparable to that of DLL3 CAR-T cells that did not overexpress other functional proteins.
  • Figures 5A-5E show a schematic flow cytometry diagram of CAR-positive detection of CAR-T cells expressing different functional structures of DLL3 by anti-human IgG (Fab) 2 antibodies, wherein Figure 5A shows the results of T cells without CAR transduction, Figure 5B shows the results for DLL3 CAR-T cells, Figure 5C shows the results for DLL3 CAR.aPDL1-T cells, Figure 5D shows the results for DLL3 CAR.mIL7-T cells, and Figure 5E shows the results for DLL3 CAR. Results for aPDL1.mIL7-T cells.
  • Fab anti-human IgG
  • Figure 6A- Figure 6E show a CAR-positive flow cytometry diagram of DLL3 CAR-T cells expressing different functional structures through DLL3 antigen detection, wherein Figure 6A shows the results of T cells without CAR transduction, and Figure 6B shows DLL3 CAR -T cell results, Figure 6C shows the results for DLL3 CAR.aPDL1-T cells, Figure 6D shows the results for DLL3 CAR.mIL7-T cells, Figure 6E shows the results for DLL3 CAR.aPDL1.mIL7-T cells the result of.
  • Figure 7A shows the results of the CAR positivity rate of DLL3 CAR-T cells with different functional structures detected by the anti-human IgG (Fab) 2 antibody detection method.
  • Figure 7B shows the results of the CAR positivity rate of DLL3 CAR-T cells with different functional structures detected by the human DLL3 antigen protein detection method.
  • the T cells after electroporation of DLL3 CAR, DLL3 CAR.aPDL1, DLL3 CAR.mIL7, and DLL3 CAR.aPDL1.mIL7 plasmids and the T cells without CAR plasmid electroporation were continuously cultured.
  • the CAR positivity rate was tested on day 11, using biotin-labeled anti-human IgG (Fab) 2 antibody and human DLL3 antigen protein as CAR-binding proteins, and then using avidin-coupled PE fluorescent dye to detect chimerism through flow cytometry.
  • Antigen receptor (CAR) expression using untransduced T lymphocytes as negative control, The changes in CAR positivity rates of DLL3 CAR-T with different structures were recorded for different days in culture.
  • Figure 8A shows the anti-human IgG (Fab) 2 antibody detection method to detect the changes in CAR positive rate of DLL3 CAR-T cells with different functional structures under different culture days
  • Figure 8B shows the human DLL3 antigen protein detection method to detect different functions Changes in CAR positivity rate of structural DLL3 CAR-T cells under different culture days.
  • Fab anti-human IgG
  • Example 4 Detection of IL7 fusion protein (mIL7) on the surface of DLL3 CAR-T cells co-expressing anti-PD-L1 and IL7 fusion protein
  • Biotin-labeled anti-human IL7 antibody is used as the binding antibody of human IL7 protein, and then avidin-coupled PE fluorescent dye is used to detect the positive rate of IL7 protein on the surface of T cell membrane through flow cytometry technology to express T lymphocytes with different functional structures.
  • the positive rate of IL7 protein on the cell membrane of the cells is shown in Table 4-1.
  • the experimental results show that the DLL3 CAR.mIL7-T and DLL3 CAR.aPDL1.mIL7-T cells expressing IL7 fusion protein of the present invention can be detected on the surface of T cells. Human IL7 protein was detected, and the expression efficiency was comparable to the CAR positive rate.
  • IL7 protein was not detected on the cell membrane surface of DLL3 CAR-T, DLL3 CAR.aPDL1-T that did not co-express IL7 fusion protein, and T cells that did not undergo CAR plasmid electroporation. .
  • Figures 9A-9E show flow cytometry diagrams of detecting IL7 protein expression on the cell membrane surface of T lymphocytes expressing different structures of DLL3 CAR by anti-human IL7 antibodies, wherein Figure 9A shows the results of T cells without CAR transduction, Figure Figure 9B shows the results for DLL3 CAR-T cells, Figure 9C shows the results for DLL3 CAR.aPDL1-T cells, Figure 9D shows the results for DLL3 CAR.mIL7-T cells, and Figure 9E shows the results for DLL3 CAR.aPDL1-T cells. Results for aPDL1.mIL7-T cells.
  • DLL3 CAR-T, DLL3 CAR.aPDL1-T, DLL3 CAR.mIL7-T, DLL3 CAR.aPDL1.mIL7-T and T cells without CAR plasmid electroporation were treated according to 5 ⁇ 10 5 CAR/T cells/mL cell density cultured for 72 hours, the cell supernatant was collected, and the small cell lung cancer cell line SHP-77-hDLL3 and SHP-77-hDLL3-hPDL1 cells were used as the target cells for the aPDL1 fusion protein binding test in the cell supernatant, where SHP The -77-hDLL3 cell line is a SHP-77 cell that highly expresses the human DLL3 protein, which was obtained by infection and screening with human DLL3 protein overexpression lentivirus (purchased from Jiman Biotechnology), while the SHP-77-hDLL3-hPDL1 cell was obtained in SHP- The human PD-L1 overexpression cell line was
  • the collected supernatant was incubated with SHP-77-hDLL3 and SHP-77-hDLL3-hPDL1 cells, and then anti-human IgG Fc-coupled APC fluorescent dye was used to detect the positivity of PD-L1 protein on the surface of tumor cells by flow cytometry. Rate.
  • Figures 10A to 10J show flow cytometry diagrams of the binding responses of CAR-T cell culture supernatants expressing different functional structures of DLL3 to different tumor cells.
  • Figures 10A-10E show a flow cytometry diagram of the binding reaction of the culture supernatant of CAR-T cells expressing different functional structures of DLL3 to SHP-77-hDLL3 cells
  • Figures 10F-10J show a schematic diagram of the binding reaction of the culture supernatant of DLL3 CAR-T cells expressing different functional structures.
  • DLL3 CAR-T, DLL3 CAR.aPDL1-T, DLL3 CAR.mIL7-T, DLL3 CAR.aPDL1.mIL7-T and T cells without CAR plasmid electroporation were treated according to 5 ⁇ 10 5 CAR/T Cells/mL cell density culture for 72 hours, collect the cell supernatant, set up 3 replicates, use anti-human IgG enzyme immunoassay kit (purchased from Novusbio) to detect the concentration of human IgG protein in the supernatant, and evaluate the concentration of aPDL1 with the concentration of human IgG protein
  • Table 5-1 The secretion content and the concentration of aPDL1 in the supernatant of T lymphocytes expressing DLL3 CARs with different functional structures are shown in Table 5-1.
  • Example 6 Conditional culture of DLL3 CAR-T cells expressing different functional structures without adding cytokines
  • DLL3 CAR-T, DLL3 CAR.aPDL1-T, DLL3 CAR.mIL7-T, DLL3 CAR.aPDL1.mIL7-T and T cells without CAR plasmid electroporation were cultured until day 10 (as T cells Sorting (day 0)), remove the cytokine human IL7 and IL15 components in the culture medium that can promote T cell expansion by centrifugation, and re-inoculate into new human IL7 and IL15 culture medium without added cytokines using the same cell density. By counting cells every 2-3 days, the viability and expansion of the cells were observed. At the end of the experiment, the CAR positivity rate of the above cells was detected by flow cytometry.
  • the experiment was set up in 2 replicate wells.
  • the cell viability of T lymphocytes expressing DLL3 CARs with different functional structures for different days cultured under factor-free conditions is shown in Table 6-1.
  • the number of viable cells for T lymphocytes expressing DLL3 CARs with different functional structures cultured under factor-free conditions for different days is shown in Table 6-1.
  • Table 6-2 the CAR positive rate of T lymphocytes expressing DLL3 CAR with different functional structures for different days cultured under factor-free conditions is shown in Table 6-3.
  • Figure 13 shows the changes in cell viability of CAR-T cells expressing DLL3 with different functional structures under cytokine-free conditions. The results of changes in the number of viable cells on different days.
  • Figure 14 shows the results of changes in the CAR positive rate of T lymphocytes expressing DLL3 CARs with different functional structures cultured under factor-free conditions on different days.
  • Table 6-1 Cell viability of T lymphocytes expressing DLL3 CARs with different functional structures cultured under cytokine-free conditions for different days.
  • Table 6-2 The number of viable cells in T lymphocytes expressing DLL3 CAR with different functional structures for different days when cultured in the presence of cytokines.
  • Example 7 In vitro cytotoxicity test of DLL3 CAR-T cells expressing different functional structures.
  • the DLL3 CAR-T, DLL3 CAR.aPDL1-T, DLL3 CAR.mIL7-T, DLL3 CAR.aPDL1.mIL7-T prepared above and T cells without CAR plasmid electroporation were used as effector cells.
  • effect-target ratio effector cells: target cells
  • a co-culture system of CAR-T cells and targeted tumor cells was established.
  • the luciferase activity method (reagent The kit (purchased from Biyuntian, Cat. No. RG028) was used to detect the tumor cell killing rate to evaluate the biological efficacy of CAR-T.
  • a control system of co-culture of non-transduced T cells and tumor cells was established, and the experiment was set up with 2 replicate wells.
  • Figure 15A- Figure 15D shows the in vitro specific killing results of DLL3 CAR-T cells expressing different functional structures on lung cancer cell lines.
  • Figure 15A shows the in vitro specific killing results of DLL3 CAR-T cells expressing different functional structures on NCI-H460 cell lines.
  • the in vitro specific killing results of the SHP-77 cell line are shown in Figure 15B.
  • Figure 15B shows the in vitro specific killing results of the SHP-77 cell line by DLL3 CAR-T cells expressing different functional structures.
  • Figure 15C shows the DLL3 CAR-T cells expressing different functional structures. In vitro specific killing results of human DLL3-positive SHP-77-hDLL3 cell line.
  • Figure 15D shows the DLL3 CAR-T cells expressing different functional structures against human DLL3-positive and human PD-L1-positive SHP-77-hDLL3- In vitro specific killing results of hPDL1 cell lines.
  • T in Figure 15A- Figure 15D represents the experimental results of T cells without CAR transduction.
  • DLL3 CAR-T cells expressing different functional structures are effective against SHP-77 (small cell lung cancer), SHP-77-hDLL3 (small cell lung cancer), and SHP-77-hDLL3.
  • -hPDL1 small cell lung cancer
  • the killing efficiency after 24 hours is 20-50%, but it is low for human DLL antigen.
  • the expressed cell line NCI-H460 had no significant killing effect. There is no significant difference in killing efficiency of DLL3 CAR-T cells expressing different functional structures at the same time.
  • DLL3 CAR-T cells expressing different functional structures can specifically kill tumor cells expressing human DLL3 antigen, and co-expression of aPDL1 fusion protein and mIL7 protein does not affect the killing effect of DLL3 CAR-T cells.
  • Table 7-1 shows the killing effect of DLL3 CAR-T cells expressing different functional structures on the above four lung cancer cell lines.
  • the specific killing detection method is carried out using a firefly luciferase reporter gene detection kit (purchased from Biyuntian).
  • Firefly luciferase is a protein with a molecular weight of approximately 61kD. In the presence of ATP, magnesium ions and oxygen Under the conditions, it can catalyze the oxidation of luciferin into oxyluciferin. During the oxidation process of luciferin, biofluorescence is emitted. Biofluorescence can be measured by a chemiluminometer (luminometer) or a liquid scintillation analyzer. Its principle is to construct the luciferase gene into a reporter gene plasmid.
  • the cells are transfected and treated with CAR-T cells or appropriate drugs.
  • the destruction of the cell membrane structure caused by cell apoptosis or necrosis will cause the enzymes in the cytoplasm to be released into the culture medium.
  • the The activity of luciferase released from cells into the culture medium can enable quantitative analysis of cytotoxicity.
  • the above-mentioned DLL3 CAR-T cells expressing different functional structures were co-cultured with four tumor cells. After 24 hours, the co-culture supernatant was harvested and secreted into the culture medium supernatant using a human IFN- ⁇ ELISA detection kit (purchased from Novusbio). Cytokine (INF- ⁇ ) content was used to evaluate the biological efficacy of CAR-T.
  • the experimental results showed that after DLL3 CAR-T expressing different functional structures killed DLL3 antigen-positive tumor cells, the release level of cytokine IFN- ⁇ was higher than that of the negative control (not shown).
  • T cells transduced with CAR at least 2 times more than DLL3 CAR-T, and compared with DLL3 CAR-T, co-expression of aPDL1 fusion protein and mIL7 protein did not affect the release of IFN- ⁇ factor from DLL3 CAR-T cells.
  • Table 7-2 shows the untransduced CAR T cells, DLL3 CAR-T cells expressing different functional structures of the present invention, IFN- ⁇ after killing four lung cancer cell lines: NCI-H460, SHP-77, SHP-77-hDLL3, SHP-77-hDLL3-hPDL1 Release level.
  • Figure 16A- Figure 16D shows the IFN- ⁇ release results after the DLL3 CAR-T cells expressing different functional structures of the present invention kill four lung cancer cells, wherein Figure 16A shows the DLL3 CAR-T expressing different functional structures and IFN- ⁇ release after co-culture of NCI-H460 cell line, Figure 16B shows IFN- ⁇ release after co-culture of DLL3 CAR-T expressing different functional structures and SHP-77 cell line, Figure 16C shows different expression IFN- ⁇ release after co-culture of DLL3 CAR-T with functional structure and SHP-77-hDLL3 cell line. Figure 16D shows co-culture of DLL3 CAR-T with different functional structure and SHP-77-hDLL3-hPDL1 cell line. IFN- ⁇ release after. T in Figures 16A-16D represents IFN- ⁇ release from T cells without CAR transduction.
  • the cytokine detection method is carried out using a human IFN- ⁇ ELISA detection kit (purchased from Novusbio), which is based on the solid phase of antigen or antibody and the enzyme labeling of antigen or antibody.
  • the antigen or antibody bound to the surface of the solid phase carrier still retains its immunological activity, and the enzyme-labeled antigen or antibody retains both its immunological activity and the activity of the enzyme.
  • the test substance (antigen or antibody) in the sample binds to the immobilized antibody or antigen. Unbound substances are removed by washing the plate, and then enzyme-labeled antigen or antibody is added.
  • the amount of enzyme that can be immobilized is related to the amount of the test substance in the sample.
  • Example 8 Test of the ability of DLL3 CAR-T cells expressing different functional structures to continuously kill tumors in vitro
  • Example 7 In order to evaluate the long-term anti-tumor enhancement effect of DLL3 CAR-T cells co-expressing aPDL1 fusion protein and mIL7 fusion protein in vitro, the present invention designed this Example 7.
  • human small cell lung cancer SHP- 77 cell line was used as the target cell.
  • effector-target ratio effector cells: target cells
  • Tumor cells were added every 3 days for repeated stimulation.
  • Figure 17A- Figure 17B shows the long-term anti-tumor effect results of DLL3 CAR-T expressing different functional structures on DLL3 expression-positive cells SHP-77, wherein Figure 17A shows the effect of DLL3 CAR-T cells expressing different functional structures on DLL3 expression The results of the change in the number of tumor cells of the long-term anti-tumor effect of SHP-77 on positive cells.
  • Figure 17B shows the change in the number of CAR-positive T cells of the long-term anti-tumor effect of SHP-77 on DLL3-expressing positive cells by DLL3 CAR-T expressing different functional structures.
  • DLL3 CAR.mIL7-T and DLL3 CAR.aPDL1.mIL7-T cells that co-expressed mIL7 fusion protein maintained good cell expansion during sustained killing, and their cell persistence was better than that of DLL3 CAR that did not co-express IL7 fusion protein.
  • -T, DLL3 CAR.aPDL1-T cells DLL3 CAR.aPDL1-T cells.
  • the inflammatory tumor microenvironment provides various factors that regulate PD-L1 expression.
  • IFN- ⁇ is mainly secreted by effector T cells and NK cells and is the most effective inducer of PD-L1 in various tumor cells.
  • IFN- ⁇ -induced PD-L1 upregulation is mediated through JAK1/2–STAT1 activation, ultimately resulting in direct binding of IFN- ⁇ regulatory factor 1 (IRF1) to the PD-L1 promoter (BMB Rep.2021Aug 31 ;54(8):403–412).
  • IRF1 IFN- ⁇ regulatory factor 1
  • CAR-T cells kill tumor cells, they release the cytokine IFN- ⁇ , which induces the upregulation of PD-L1 in tumor cells and further combines with PD-1 of CAR-T cells, causing CAR-T cell exhaustion.
  • the DLL3 CAR-T cells co-expressing aPDL1 fusion protein in the present invention can secrete aPDL1 fusion protein and can bind to the PD-L1 protein of tumor cells. Therefore, based on Example 7, the PD of SHP77 tumor cells after killing by CAR-T cells -L1 protein was analyzed by flow cytometry.
  • the experimental results showed that when DLL3 CAR-T cells expressing different functional structures killed SHP-77 cells, the cell membrane of SHP-77 cells showed that the expression of PD-L1 protein was significantly up-regulated, while the co-expression of aPDL1 fusion
  • the aPDL1 fusion protein secreted by DLL3 CAR.aPDL1-T and DLL3 CAR.aPDL1.mIL7-T cells can bind to the PD-L1 protein of SHP-77 cells, reduce the detection value of PD-L1 protein, and thereby block PD-1 and the PD-L1 pathway.
  • Table 8-1 shows the PD-L1 expression results of the remaining tumor cells after killing SHP-77 tumor cells by T cells without CAR transduction and DLL3 CAR-T cells expressing different functional structures of the present invention.
  • Figure 18 shows the PD-L1 protein expression results of SHP-77 cells after sustained killing by DLL3 CAR-T cells expressing different functional structures.
  • aPDL1 fusion protein secreted by DLL3 CAR-T cells co-expressing aPDL1 fusion protein in the present invention can bind to the PD-L1 protein of tumor cells, thereby blocking the PD-1 and PD-L1 pathways.
  • DLL3 CAR-T cells co-expressing mIL7 fusion protein can enhance the expansion ability and persistence of CAR-T cells.
  • DLL3 CAR-T cells that co-express aPDL1 fusion protein and mIL7 fusion protein retain the functions of the above two structures and enhance the ability of DLL3 CAR-T cells to continuously kill tumors.
  • Example 7 an immune-deficient mouse drug efficacy model with human small cell lung cancer tumor cell load was established to evaluate the drug efficacy of DLL3 CAR-T cells co-expressing aPDL1 fusion protein and mIL7 fusion protein in mice. effect.
  • female NCG mice purchased from Jicui Yaokang
  • the SHP-77 cells were inoculated on the 6th day (tumor volume was 50-60 mm3).
  • T cells T cells without CAR transduction
  • DLL3 CAR-T DLL3 CAR-T cells without co-expression of aPDL1 fusion protein and mIL7 fusion protein
  • co-expression of aPDL1 fusion protein protein and mIL7 fusion protein DLL3 CAR-T cell (DLL3 CAR.aPDL1.mIL7-T) group, in which the T cell group was administered at a dose of 5 ⁇ 10 6 T cells/mouse, and the DLL3 CAR-T cell group was administered at a dose of 5 ⁇ 10 6 T cells/mouse. The dose of ⁇ 10 6 CAR-T cells/mouse was administered.
  • the DLL3 CAR.aPDL1.mIL7-T cell group was administered at a dose of 2 ⁇ 10 6 CAR-T cells/mouse.
  • the number of animals in all condition groups was 5. Only. After administration, tumors were measured twice a week; tumor growth curves were drawn.
  • Figure 19 shows the results of tumor size changes in small cell lung cancer SHP-77 tumor-bearing NCG mice after administration of DLL3 CAR-T cells, DLL3 CAR.aPDL1.mIL7-T cells, and non-CAR-transduced T cells.

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Abstract

本发明涉及一种靶向DLL3的抗体、抗原结合片段，靶向DLL3的第四代CAR及其载体的制备和应用。所述抗DLL3抗体或其抗原结合片段对DLL3以及表达DLL3的细胞具有良好的亲和力。所述靶向人DLL3抗原的第四代CAR结构包含免疫抑制分子表达元件、细胞膜型白介素和/或分泌型趋化因子的表达元件，该CAR结构转染的细胞在持续性杀伤中保持良好的细胞扩增，显著增强DLL3 CAR-T细胞的抗肿瘤能力。本申请还提供了编码所述抗体或其抗原结合片段和编码靶向DLL3抗原的CAR的核酸、表达盒、载体、含有所述表达盒、载体或核酸的细胞、表达盒、药物组合物和试剂盒，以及提供用于检测或诊断、治疗或改善与DLL3相关的疾病的应用，在医药领域有广阔的前景。

Description

一种靶向人DLL3抗原的第四代CAR及其载体的制备和应用 技术领域
本发明涉及生物医学或生物制药技术领域，尤其涉及一种共表达免疫抑制分子和细胞因子的靶向人DLL3抗原的CAR结构，及其制备方法和用于制备细胞药物(如T细胞、NK细胞、巨噬细胞等具有免疫功能的人体细胞)、治疗、预防疾病的用途。
背景技术
近年来，由于饮食、环境、人口的老龄化等因素，全球癌症发病率不断增长，癌症被认为是世界上每个国家的主要死亡原因和延长预期寿命的重要障碍。根据国际癌症研究机构(IARC)发布的2020年全球最新癌症负担数据，中国已经成为了名副其实的癌症大国，不论是新发人数还是死亡人数，中国都位居全球第一。其中肺癌连续10年位居我国恶性肿瘤之首，在2020年中国肺癌死亡人数有近71.5万，占癌症死亡人数的23.8％。小细胞肺癌(SCLC)是肺癌中的一种，根据NCCN指南，约占所有肺癌的15％，具有进展快、高复发率、早期转移、预后差的特点，5年生存率低于5％，且治疗手段有限。传统放化疗只能短期获益，暂无明显延长患者生存时间的有效手段。
嵌合抗原受体(Chimeric antigen receptor，CAR)修饰T细胞(CAR-T细胞治疗技术)是近年来发展非常迅速的一种过继性免疫细胞技术。通过基因改造技术，其效应T细胞的靶向性、杀伤活性及持久性均优于常规应用的免疫细胞，并可克服肿瘤局部免疫抑制微环境和打破宿主免疫耐受状态，是肿瘤免疫细胞治疗领域中新的靶向治疗方式。
人DLL3蛋白(δ样配体，Delta-Like Ligand 3，DLL3)是由619个氨基酸组成的单次跨膜蛋白，属于Notch配体家族。它是一种高度肿瘤选择性的细胞表面靶点，在大多数小细胞肺癌(SCLC)和类癌亚组中高表达(Lung Cancer；135:73-79；2019)，但在正常肺癌组织及癌旁组织中不表达。在一项研究中，对1073例SLCL患者的独立肿瘤标本研究显示，DLL3阳性表达(≥25％)达到85％，DLL3高表达(≥75％)达到68％(Lung Cancer；147:237-243；2020)。靶向人DLL3的嵌合抗原受体T细胞可以通过识别肿瘤表面DLL3抗原从而刺激T细胞对肿瘤细胞进行特异性杀伤。
目前全球已有多款CAR-T产品经FDA批准上市，治疗多种不同类型的血液系统恶性肿瘤。而在占比超90％以上的实体瘤领域，CAR-T疗法则进展缓慢，CAR-T的实体瘤治疗主要有以下几大问题：1、缺乏有效靶点；2、CAR-T的转运和浸润；3、肿瘤微环境的免疫抑制；4、内源性T细胞抑制信号。
针对以上，本发明通过优化CAR载体结构，赋予免疫细胞更多功能，尤其是共表达免疫抑制分子和/h或因子的靶向人DLL3抗原的CAR结构，有望对DLL3抗原阳性肿瘤患者的治疗带来希望。
发明内容
为解决提供更多抗癌药物，特别是提供针对高表达DLL3的癌症的药物的技术问题，例如治疗肺癌、黑色素瘤、甲状腺髓样癌、胶质母细胞瘤、前列腺癌、神经内分泌癌中的一种或者多种的药物，尤其是肺癌，特别是小细胞肺癌的药物，本发明提供一种以DLL3为靶点的嵌合抗原受体(CAR)，包含所述CAR的免疫细胞，包括但不限于T细胞。本发明还提供了编码所述CAR的核酸；含有所述核酸的表达盒、载体、细胞；含有所述CAR、所述核酸、所述表达盒、所述载体、所述细胞的药物组合物；含有所述CAR、所述核酸、所述表达盒、所述载体、所述细胞、所述药物组合物的试剂盒；所述CAR、包含所述CAR的免疫细胞、所述核酸、所述表达盒、所述载体、所述细胞、所述药物组合物在预防、治疗、检测或诊断与DLL3相关的疾病的应用，或在制备预防和/或治疗癌症或肿瘤的药物或制剂的用途，所述与DLL3相关的疾病是DLL3高表达疾病，进一步所述疾病是DLL3高表达癌症或肿瘤，再进一步，所述癌症或肿瘤选自肺癌、黑色素瘤、甲状腺髓样癌、胶质母细胞瘤、前列腺癌、神经内分泌癌中的一种或多种；再进一步所述癌症或肿瘤是肺癌， 特别是小细胞肺癌。本发明还提供一种制备工程化免疫细胞的方法。
本发明第一个方面提供一种抗DLL3抗体或其抗原结合片段，所述抗体或其抗原结合片段包含下述3个轻链互补决定区和/或3个重链互补决定区，所述抗体或其抗原结合片段的3个轻链互补决定区包含如SEQ ID NO:22所示LCDR1,如SEQ ID NO:23所示LCDR2,如SEQ ID NO:24所示LCDR3；和/或3个重链互补决定区包含如SEQ ID NO:18所示HCDR1，如SEQ ID NO:19所示的HCDR2,如SEQ ID NO:20所示的HCDR3。
进一步的，所述抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含与SEQ ID NO:21所示的氨基酸序列具有少95％、96％、97％、98％、99％或100％同一性的轻链可变区(VL)，和与SEQ ID NO:17所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的重链可变区(VH)。
进一步的，所述抗体包含与SEQ ID NO:25所示序列具有少95％、96％、97％、98％、99％或100％同一性的重链恒定区，和包含与SEQ ID NO:26所示序列具有少95％、96％、97％、98％、99％或100％同一性的轻链恒定区。
本发明第二个方面，一种靶向DLL3抗原的嵌合抗原受体(CAR)，所述CAR包含DLL3抗原结合结构域、跨膜域和胞内信号转导结构域，其中所述DLL3抗原结合结构域是scFv,其中所述scFv为权利要求1-2任一项所述抗体或其抗原结合片段。
进一步的，所述靶向DLL3抗原的嵌合抗原受体，其特征在于，所述CAR还包含铰链区、信号肽和共刺激信号域中的一种或多种；
优选地，所述跨膜结构域为CD8跨膜区，所述铰链区为CD8铰链区，所述细胞内信号传导结构域为CD3ζ胞内信号域，所述信号肽为CD8α信号肽，或所述共刺激信号域为4-1BB或者CD28共刺激信号域。
进一步的，所述靶向DLL3抗原的嵌合抗原受体，其特征在于，所述CAR从N端到C端依次包含CD8α信号肽、DLL3抗体scFv VH-linker-DLL3抗体scFv VL、CD8铰链区、CD8跨膜区、4-1BB共刺激信号和CD3ζ胞内信号域。
进一步的，所述靶向DLL3抗原的嵌合抗原受体，其特征在于，
所述CD8α信号肽包含与SEQ ID NO:27所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；
所述CD8铰链区及跨膜区包含与SEQ ID NO:28所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；
所述4-1BB共刺激信号域包含与SEQ ID NO:29所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；或者
所述CD3ζ胞内信号域包含与SEQ ID NO:30所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；
优选地，所述CAR的氨基酸序列如SEQ ID NO:32所示。
进一步的，所述的靶向DLL3抗原的嵌合抗原受体，其特征在于，所述CAR包含如下元件i)和/或ii)：
i)免疫抑制分子表达元件；
ii)细胞膜型白介素和/或分泌型趋化因子的表达元件，
优选的，所述免疫抑制分子选自：PD1和PDL1中的一个或者多个；所述细胞膜型白介素选自细胞膜型IL2细胞因子、细胞膜型IL4细胞因子、细胞膜型IL7细胞因子、细胞膜型IL9细胞因子、细胞膜型IL10细胞因子、细胞膜型IL15细胞因子、、细胞膜型IL18细胞因子、细胞膜型IL21细胞因子、细胞膜型IL23细胞因子、细胞膜型IL24细胞因子、细胞膜型IL36细胞因子中的一个或者多个；所述分泌型趋 化因子选自分泌型CCL1趋化因子、分泌型CCL2趋化因子、分泌型CCL3趋化因子、分泌型CCL5趋化因子、分泌型CCL7趋化因子、分泌型CCL15趋化因子、分泌型CCL16趋化因子、分泌型CCL19趋化因子、分泌型CCL20趋化因子、分泌型CCL21趋化因子、分泌型CXCL4趋化因子、分泌型CXCL9趋化因子、分泌型CXCL10趋化因子、分泌型CXCL11趋化因子、分泌型XCL1趋化因子中的一个或者多个。
进一步的，所述的靶向DLL3抗原的嵌合抗原受体，包含抗PDL1蛋白表达元件和/或细胞膜型IL7细胞因子表达元件；
优选的，所述抗PDL1表达元件依次包含kappa前导信号肽、抗PD-L1抗体scfv、连接肽1和人IgG CH2CH3片段；所述细胞膜型IL7细胞因子表达元件依次包含人IL-7细胞因子片段、连接肽2、CD8跨膜区；
更优选的，所述抗PDL1表达元件中的kappa前导信号肽包含与SEQ ID NO:33所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；抗PDL1抗体scfv包含与SEQ ID NO:34所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；连接肽1的氨基酸序列为GGGS；人IgG CH2CH3片段包含SEQ ID NO:35所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；所述细胞膜型IL7细胞因子表达元件中的人IL-7细胞因子片段包含与SEQ ID NO:36所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；连接肽2氨基酸序列为SGGGSGGGGSGGGGSGGGGSGGGSLQ；CD8跨膜区氨基酸序列包含与SEQ ID NO:37所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；所述抗PDL1表达元件和细胞膜型IL7细胞因子表达元件两者以2A肽连接，所述2A肽序列如SEQ ID NO:38所示。
在本发明的一个优选的实例中，所述的靶向DLL3抗原的嵌合抗原受体包含抗PDL1蛋白表达元件，进一步的，所述CAR的氨基酸序列如SEQ ID NO:43所示。
在本发明的一个优选的实例中，所述的靶向DLL3抗原的嵌合抗原受体包含细胞膜型IL7细胞因子表达元件，进一步的，所述CAR的氨基酸序列如SEQ ID NO:44所示。
在本发明的一个优选的实例中，所述的靶向DLL3抗原的嵌合抗原受体包含抗PDL1蛋白表达元件和细胞膜型IL7细胞因子表达元件，进一步的，所述CAR的氨基酸序列如SEQ ID NO:45所示。
本发明第三个方面提供一种核酸，其编码所述的抗DLL3抗体或其抗原结合片段或编码所述的靶向DLL3抗原的嵌合抗原受体。
本发明第四个方面提供一种表达盒，其包含所述的核酸。
本发明第五个方面提供一种载体，其含所述核酸或所述表达盒。本发明的第六方面提供了一种核酸，其编码所述抗DLL3抗体或其抗原结合片段或者CAR。
本发明的第六个方面提供了一种表达盒，其包含所述核酸。
本发明的第七个方面提供了一种载体，其包含编码所述抗DLL3抗体或其抗原结合片段的核酸、CAR的核酸、或所述表达盒。所述载体可用于表达所述抗DLL3抗体或其抗原结合片段或者表达所述CAR。优选地，所述载体可以是病毒载体；优选地，所述病毒载体包含但不限于慢病毒载体、腺病毒载体、腺相关病毒载体或逆转录病毒载体等；优选地，所述载体可以是非病毒载体；优选地，所述载体可以是哺乳细胞表达载体；优选地，所述表达载体可以是细菌表达载体；优选地，所述表达载体可以是真菌表达载体。
本发明的第八方面提供了一种细胞，所述细胞包括所述核酸、或所述表达盒、或所述载体，所述细胞可表达所述抗DLL3抗体或其抗原结合片段或者所述CAR。优选地，所述细胞为细菌细胞；优选地，所述细菌细胞为大肠杆菌细胞等；优选地，所述细胞为真菌细胞；优选地，所述真菌细胞为酵母细胞；优选地，所述酵母细胞为毕赤酵母细胞等；优选地，所述细胞为哺乳动物细胞；优选地，所述哺乳动物细胞为中国仓鼠卵巢细胞(CHO)、人胚胎肾细胞(293)、B细胞、T细胞、DC细胞或NK细胞等。优选地，所述细胞为 工程化的免疫细胞；更优选地，所述工程化的免疫细胞为T细胞；最优选地，所述T细胞为原代来源T细胞或者iPSC分化来的T细胞，所述IPSC分化来的T细胞为γδT细胞、DNT细胞或NKT细胞。
本发明的第九个方面提供了一种药物组合物，其包含所述CAR、核酸、表达盒、载体或细胞，优选地，所述药物组合物还包含药学上可接受的载体，优选地，所述药学上可接受的载体包括以下中的一种或多种：药学上可接受的溶剂、分散剂、附加剂、塑形剂、药物辅料。
本发明的第十个方面提供了一种试剂盒，其包含本发明所述抗DLL3抗体或其抗原结合片段、所述CAR，或包含编码CAR的核酸、或所述表达盒。
本发明的第十一个方面提供了所述抗DLL3抗体或其抗原结合片段、所述CAR、核酸、表达盒、载体或细胞在制备治疗或预防疾病的药物组合物中的应用。
本发明的第十二个方面提供了所述抗DLL3抗体或其抗原结合片段、所述CAR或核酸或表达盒在制备诊断、检测试剂盒中的应用。
本发明的第十三个方面提供了一种治疗或预防疾病的方法，包括将本发明的所述抗DLL3抗体或其抗原结合片段、所述CAR、核酸、表达盒、载体、细胞或药物组合物给予有需要的受试者。
本发明的第十四个方面提供了一种诊断、检测的方法，包括将本发明的所述抗DLL3抗体或其抗原结合片段、所述CAR、核酸、表达盒、试剂盒或药物组合物给予有需要的受试者或样本。
本发明的第十五个方面提供了所述抗DLL3抗体或其抗原结合片段、所述CAR、核酸、表达盒、载体、细胞或药物组合物用于治疗、预防疾病的用途。
本发明的第十六个方面提供了所述抗DLL3抗体或其抗原结合片段、所述CAR、核酸、表达盒、试剂盒、或药物组合物用于检测、诊断的用途。
本发明的第十七个方面提供了所述抗DLL3抗体或其抗原结合片段、所述CAR，所述核酸、或所述表达盒，或所述载体、或所述药物组合物用于预防、治疗、检测或诊断与DLL3相关的疾病的应用。
在本发明的方案中，所述与DLL3相关的疾病是DLL3高表达疾病；优选地，所述疾病是DLL3高表达癌症或肿瘤；更优选地，所述癌症或肿瘤选自肺癌、黑色素瘤、甲状腺髓样癌、胶质母细胞瘤、前列腺癌、神经内分泌癌中的一种或多种；最优选地，所述癌症是肺癌，特别是小细胞肺癌。
本发明的第十八个方面提供了一种制备工程化免疫细胞的方法，其特征在于，包括以下步骤：
(1)提供一待改造的免疫细胞；和
(2)将所述核酸、或所述表达盒、或所述载体导入到所述免疫细胞。
优选地，所述免疫细胞为T细胞；更优选地，所述T细胞为原代来源T细胞或者iPSC分化来的T细胞，所述iPSC分化来的T细胞为γδT细胞、DNT细胞或NKT细胞。
本发明的第十九个方面还提供DLL3抗体或其抗原结合片段在制备抗体-药物偶联物(ADC)中的应用。
本发明第二十个方面提供一种抗DLL3抗体-药物偶联物，所述抗DLL3抗体-药物偶联物包括(a)本发明的抗DLL3抗体或其抗原结合片段,和(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在一优选例中，所述药物选自下组：化疗药物、放疗药物、激素治疗药物或免疫治疗药物。
本发明提供的靶向人DLL3抗原的第四代CAR及其载体，尤其是共表达抗PD1或PD-L1融合蛋白、和/或白介素融合蛋白的靶向人DLL3抗原的CAR载体具有以下的一种或多种优势：
1、本发明提供的抗DLL3抗体或其抗原结合片段对人DLL3蛋白，以及表达DLL3的细胞均具有良好的亲和力。
2、本发明构建的表达DLL3 CAR的重组质粒(DLL3 CAR)、表达DLL3 CAR和抗PD-L1融合蛋白的重组质粒(DLL3 CAR.aPDL1)、表达DLL3 CAR和细胞膜型IL7融合蛋白的重组质粒(DLL3 CAR.mIL7)和共表达DLL3 CAR、抗PD-L1 scfv融合蛋白及细胞膜IL7融合蛋白两种功能结构的重组质粒(DLL3  CAR.aPDL1.mIL7)的四种质粒均可用于制备CAR-T细胞，CAR阳性率范围在10％-60％。
3.采用本发明构建的重组质粒转染的细胞获得表面稳定表达抗PD-L1蛋白的DLL3 CAR-T细胞(DLL3 CAR.aPDL1-T细胞)，稳定表达mIL7蛋白的DLL3 CAR-T细胞(DLL3 CAR.mIL7-T细胞)，和稳定共表达抗PD-L1及IL7融合蛋白的DLL3 CAR-T细胞(DLL3 CAR.aPDL1.mIL7-T细胞)。
4.本发明构建的共表达mIL7融合蛋白的CAR-T细胞能够增强CAR-T细胞的活力及持续性，mIL7融合蛋白主要作用于T细胞自身CAR阳性细胞群体，引发自身CAR阳性细胞群体扩增和保持活性。
5.表达不同功能结构的DLL3 CAR-T细胞能够特异性的杀伤人DLL3抗原表达的肿瘤细胞，且共表达aPDL1融合蛋白和mIL7蛋白未影响DLL3 CAR-T细胞杀伤效果。
6.本发明构建的表达不同功能结构的DLL3 CAR-T杀伤DLL3抗原阳性肿瘤细胞后细胞因子IFN-γ释放水平高于阴性对照(未转导CAR的T细胞)至少2倍以上，且对比DLL3 CAR-T，共表达aPDL1融合蛋白和mIL7蛋白未影响DLL3 CAR-T细胞IFN-γ因子释放。
7.本发明构建的共表达mIL7融合蛋白的DLL3 CAR.mIL7-T细胞、DLL3 CAR.aPDL1.mIL7-T细胞对肿瘤细胞的清除作用明显优于未共表达IL7融合蛋白的DLL3 CAR-T细胞、DLL3 CAR.aPDL1-T细胞；而且共表达mIL7融合蛋白的DLL3 CAR.mIL7-T细胞、DLL3 CAR.aPDL1.mIL7-T细胞在持续性杀伤中保持良好的细胞扩增，其细胞持续性优于未共表达IL7融合蛋白的DLL3 CAR-T、DLL3 CAR.aPDL1-T细胞。
8.本发明中共表达aPDL1融合蛋白的DLL3 CAR-T细胞分泌的aPDL1融合蛋白能够结合肿瘤细胞的PD-L1蛋白，从而阻断PD-1与PD-L1通路。而共表达mIL7融合蛋白的DLL3 CAR-T细胞能够增强CAR-T细胞的扩增能力及持续性。共表达aPDL1融合蛋白及mIL7融合蛋白的DLL3 CAR-T细胞保留了以上两种结构的功能，增强了DLL3 CAR-T细胞持续杀伤肿瘤的能力。
9.在小细胞肺癌肿瘤细胞负荷的免疫缺陷型小鼠药效模型中，DLL3 CAR-T细胞、DLL3 CAR.aPDL1.mIL7-T细胞均对小鼠体内SHP-77肿瘤有抑制作用；共表达aPDL1融合蛋白及mIL7融合蛋白的DLL3 CAR-T细胞显示出在低剂量下更优的肿瘤抑制效果，说明共表达aPDL1融合蛋白及mIL7融合蛋白能够显著增强DLL3 CAR-T细胞的抗肿瘤能力。
10.本发明在二代CAR结构scfv抗原识别区、4-1BB共刺激区、CD3ζ信号的基础上，构建四代CAR及其载体，共表达抗PD-L1 scfv融合蛋白(aPDL1)和/或及细胞膜IL7融合蛋白(mIL7)两种功能结构,其中抗PD-L1 scfv融合蛋白能阻断PD-1/PD-L1信号通路，降低T细胞耗竭，维持T细胞稳定，细胞膜IL7融合蛋白增强T细胞活力和扩增能力，从而解决CAR-T细胞免疫抑制肿瘤微环境、体内持续性差等的问题。
附图说明
图1为实施例1中DLL3蛋白免疫小鼠血清滴度中500x，2500x，12500x，以及62500x代表血清稀释比例。
图2为实施例1抗体与人DLL3-His蛋白的ELISA结合活性结果。
图3为实施例1中抗体与CT26-hDLL3细胞的结合活性结果。
图4A-4C为实施例2中基因片段和载体结构示意图，其中图4A为DLL3 CAR、DLL3 CAR.aPDL1、DLL3 CAR mIL7、DLL3 CAR.aPDL1.mIL7基因片段结构；图4B为重组质粒pRRLSIN-DLL3 CAR(PB DLL3 CAR)结构示意图；图4C为重组质粒pRRLSIN-DLL3 CAR.aPDL1.mIL7(PB DLL3 CAR.aPDL1.mIL7)结构示意图。
图5A-图5E为实施例3中通过抗人IgG(Fab)2抗体检测表达不同功能结构DLL3 CAR-T细胞的CAR阳性流式示意图，其中图5A示出了未转导CAR的T细胞结果，图5B示出了DLL3 CAR-T细胞的结果，图5C示出了DLL3 CAR.aPDL1-T细胞的结果，图5D示出了DLL3 CAR.mIL7-T细胞的结果，图5E示出了DLL3 CAR.aPDL1.mIL7-T细胞的结果。
图6A-图6E为实施例3中通过DLL3抗原检测表达不同功能结构DLL3 CAR-T细胞的CAR阳性流式示意图，其中图6A示出了未转导CAR的T细胞结果，图6B示出了DLL3 CAR-T细胞的结果，图6C示出了DLL3 CAR.aPDL1-T细胞的结果，图6D示出了DLL3 CAR.mIL7-T细胞的结果，图6E示出了DLL3 CAR.aPDL1.mIL7-T细胞的结果。
图7A为实施例3中抗人IgG(Fab)2抗体检测方法检测不同功能结构DLL3 CAR-T细胞的CAR阳性 率结果；图7B为实施例3中人DLL3抗原蛋白检测方法检测不同功能结构DLL3 CAR-T细胞的CAR阳性率结果。
图8A为实施例3中抗人IgG(Fab)2抗体检测方法检测不同功能结构DLL3 CAR-T细胞在不同培养天数下CAR阳性率的变化结果；图8B为实施例3中人DLL3抗原蛋白检测方法检测不同功能结构DLL3 CAR-T细胞在不同培养天数下CAR阳性率的变化结果。
图9A-图9E为实施例4中通过抗人IL7抗体检测表达不同结构DLL3 CAR的T淋巴细胞的细胞膜表面IL7蛋白表达的流式示意图，其中图9A示出了未转导CAR的T细胞结果，图9B示出了DLL3 CAR-T细胞的结果，图9C示出了DLL3 CAR.aPDL1-T细胞的结果，图9D示出了DLL3 CAR.mIL7-T细胞的结果，图9E示出了DLL3 CAR.aPDL1.mIL7-T细胞的结果。
图10A-图10J为实施例5中了表达不同功能结构DLL3 CAR-T细胞培养上清对不同肿瘤细胞结合反应的流式示意图。其中图10A-10E示出了表达不同功能结构DLL3 CAR-T细胞培养上清对SHP-77-hDLL3细胞结合反应的流式示意图，图10F-10J示出了表达不同功能结构DLL3 CAR-T细胞培养上清对SHP-77-hDLL3-hPDL1细胞结合反应的流式示意图。
图11为实施例5中表达不同功能结构的DLL3 CAR-T细胞培养上清中人IgG蛋白(aPDL1)浓度测试结果。
图12为实施例6中表达不同功能结构的DLL3 CAR-T细胞在无因子条件培养下不同天数细胞活率变化结果。
图13为实施例6中表达不同功能结构的DLL3 CAR-T细胞在无因子条件培养下不同天数活细胞数量变化结果。
图14为实施例6中表达不同功能结构的DLL3 CAR的T细胞在无因子条件培养下不同天数CAR阳性率变化结果。
图15A-图15D为实施例7中表达不同功能结构的DLL3 CAR-T细胞对肺癌细胞株的体外特异性杀伤结果，图15A示出了表达不同功能结构的DLL3 CAR-T细胞对NCI-H460细胞株的体外特异性杀伤结果，图15B示出了表达不同功能结构的DLL3 CAR-T细胞对SHP-77细胞株的体外特异性杀伤结果，图15C示出了表达不同功能结构的DLL3 CAR-T细胞对人DLL3阳性SHP-77-hDLL3细胞株的体外特异性杀伤结果，图15D示出了表达不同功能结构的DLL3 CAR-T细胞对人DLL3阳性、人PD-L1阳性的SHP-77-hDLL3-hPDL1细胞株的体外特异性杀伤结果，图15A-图15D中T代表未转导CAR的T细胞实验结果。
图16A-图16D为实施7中表达不同功能结构的DLL3 CAR-T细胞对四种肺癌细胞杀伤后IFN-γ释放结果，其中图16A示出了表达不同功能结构的DLL3 CAR-T与NCI-H460细胞株共培养后的IFN-γ释放，图16B示出了表达不同功能结构的DLL3 CAR-T与SHP-77细胞株共培养后的IFN-γ释放，图16C示出了表达不同功能结构的DLL3 CAR-T与SHP-77-hDLL3细胞株共培养后的IFN-γ释放，图16D示出了表达不同功能结构的DLL3 CAR-T与SHP-77-hDLL3-hPDL1细胞株共培养后的IFN-γ释放，图16A-16D中T代表未转导CAR的T细胞的IFN-γ释放。
图17A-图17B为实施例8中表达不同功能结构的DLL3 CAR-T对DLL3表达阳性细胞SHP-77长期抗肿瘤作用结果，其中图17A示出了表达不同功能结构的DLL3 CAR-T细胞对DLL3表达阳性细胞SHP-77长期抗肿瘤作用的肿瘤细胞数量变化结果，图17B示出了表达不同功能结构的DLL3 CAR-T对DLL3表达阳性细胞SHP-77长期抗肿瘤作用的CAR阳性T细胞数量变化结果。
图18为实施例8中SHP-77细胞在表达不同功能结构的DLL3 CAR-T细胞持续杀伤后PD-L1蛋白表达结果。
图19为实施例9中DLL3 CAR-T细胞、DLL3 CAR.aPDL1.mIL7-T细胞及未转导CAR的T细胞给药后小细胞肺癌SHP-77肿瘤荷瘤NCG小鼠的肿瘤大小变化结果。
具体实施方式
下面结合具体实施例，进一步阐述本发明。所描述的实施例是本发明一部分实施例，而不是全部的实施例。应理解，举出以下实施例是为了向本发明所属技术领域的一般专业人员就如何利用本发明之方法和组合物提供一个完整的公开和说明，并非用于限制本发明的范围。基于本发明中的实施例，本领域 普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：抗DLL3抗体产生与抗体的分子表征
1.1方法材料
表1-1使用的免疫蛋白和小鼠
将DLL3-Fc、DLL3-His两种标签的DLL3抗原蛋白，采用腹部皮下注射的方式免疫小鼠，首免使用弗氏完全佐剂乳化抗原，二次免疫-四次免疫使用弗氏不完全佐剂乳化抗原。使用表1蛋白免疫小鼠后，检测小鼠血清滴度结果如图1所示，图1展示的DLL3蛋白免疫小鼠血清滴度中500x，2500x，12500x，以及62500x代表血清稀释比例。
1.2噬菌体库的建立
处死小鼠，解剖取出脾脏，把脾脏用注射器胶塞研磨破碎并用滤网过滤，把滤过的脾细胞冷冻备，提取RNA后获得cDNA，噬菌体库的建立依据常规方法进行。构建库的库容数据如表1-2所示。
表1-2毒株免疫小鼠构建噬菌体库库容
1.3以两种方法进行筛选
1.3.1平板筛选，用DLL3-His蛋白包被平板。第二天加入噬菌体库孵育2h，洗涤4-10次后用洗脱缓冲液洗脱特异性结合的噬菌体。
1.3.2磁珠筛选，将DLL3-Fc蛋白按照试剂盒步骤进行生物素化，再与Thermo的磁珠结合，经BSA封闭后与噬菌体库孵育2h，洗涤4-10次后用Elution Buffer洗脱特异性结合的噬菌体。筛选获得的抗体克隆及来源库见表1-3。
表1-3活性抗体来源
1.4.完整抗DLL3抗体分子的构建与生产
通过常规的分子生物学技术PCR(2×Phanta Max Master Mix厂家：Vazyme货号：P515-P1-AA批号：7E351H9)扩增抗体可变区基因，通过同源重组分别将抗体重链可变区基因连接入带有抗体重链恒定区序列的核酸序列的载体pCDNA3.4(Life Technology)，将抗体轻链可变区基因连接入带有抗体轻链恒定区序列的核酸序列的载体pCDNA3.4。本申请实施例中构建阳性克隆IgG1的各抗体的可变区序列参见表1-4，重 链和轻链恒定区序列见表1-5。
将测序后的阳性克隆提取质粒后共转染进入HEK293细胞在37℃\8％CO2\125rpm摇床中培养，瞬时表达7天后上清通过Protein A亲和层析，纯化获得抗体，并通过UV280结合理论消光系数确定抗体浓度。
表1-4：抗体可变区氨基酸序列
注：VL、VH中下划线部分为CDR，分析系统为IMGT系统
表1-5重链和轻链恒定区序列
1.5 Anti-DLL3单克隆抗体分子的表征
1.5.1 ELISA检测抗体与人DLL3-His蛋白的结合
用pH 9.6CBS稀释人DLL3-His蛋白至0.1μg/mL包被酶标板,100μL/孔，4℃孵育过夜；洗板后用脱脂奶粉封闭。洗板后每孔加入PBST梯度稀释好的抗体100μL，37℃孵育1h；洗板后加入PBST稀释的HRP标记的羊抗人IgG(H+L)抗体，100μL/孔，37℃孵育1h。洗板后每孔加入100μL TMB显色，10min后每孔加入50μL 2M H2SO4终止显色，用酶标仪读取OD450，图2为抗体与人DLL3-His蛋白的ELISA结合活性，结果显示抗体ID为229、244和359的3个抗体与人DLL3-His蛋白均表现出较佳的结合活性。图2中的Isotype为自制的其他靶点的无关IgG1抗体，作为空白对照。Isotype未显示结合活性，以抗体浓度为0.1μg/mL为例，Isotype OD450＝0.127，抗体组OD450为2.8-4.5，尤其是编码为359的抗体其OD450值为Isotype组的22倍以上。
1.5.2流式细胞仪检测抗体与CT26-hDLL3细胞的结合
用PBS将对数生长期的CT26-hDLL3细胞(康源博创，KC-1129)稀释至2E6/mL，将梯度稀释的抗体和细胞混合：100μL体系＝50μL抗体+50μL细胞，4℃静置孵育1h。离心弃上清后，用PBS重悬洗涤细胞，加入100μL/w二抗FITC anti-human IgG Fc，混合均匀，避光4℃孵育0.5h。用PBS洗涤细胞后用100μL/孔PBS重悬细胞，用流式细胞仪检测。各抗体与细胞的结合活性曲线如图3所示，图3展示了抗体与CT26-hDLL3细胞的结合活性，图3中的Isotype为自制的其他靶点的无关IgG1抗体，作为空白对照。Isotype未表现出结合活性，以抗体浓度为10μg/mL为例，Isotype的MFI为10120，抗体组MFI为26276-78408，抗体组MFI为Isotype的2.6-7.7倍，EC50值见表1-6。抗体DL3-BA359和CT26-hDLL3细胞特异结合，且结合活性较高，结合EC50为0.066，优于抗体编号为DL3-BA244、DL3-BA229的抗体。
表1-6各抗体与CT26-hDLL3细胞结合的EC50
1.5.3检测抗体与hDLL3-His蛋白的亲和力
将人DLL3-His蛋白用1x HBS-EP+Buffer 2倍梯度稀释5个浓度，分别为50nM、25nM、12.5nM、6.25nM和3.125nM。用1x HBS-EP+稀释抗体样品至2μg/mL，用ProA芯片捕获抗体样品，设置仪器捕获时间为70s，流速10μL/min；分析物：结合60s，流速30μL/min，解离600s；再生：用pH 1.5的10mM Glycine-HCl buffer再生30s，流速30μL/min，Startup 3次。使用1:1 binding模型(BIAcore Insight Evaluation Software version 2.0.15.12933)计算平衡解离常数(KD)。各抗体的亲和力结果如表1-7所示，所有抗体的KD均小于1.0E-09展现出较高的亲和力，其中编号为BA359的抗体 展示出最高的亲和力。
表1-7各抗体与人DLL3-His蛋白的亲和力
实施例2构建重组质粒
2.1靶向DLL3嵌合抗原受体(CAR)基因片段制备
本发明按以下编码基因的顺序设计靶向DLL3嵌合抗原受体基因片段：CD8α信号肽、DLL3 antibody scFv VH-linker-DLL3 antibody scFv VL、CD8铰链区、CD8跨膜区以及4-1BB共刺激信号和CD3ζ胞内信号域，所述结构委托南京金斯瑞生物科技有限公司通过基因合成技术合成，使表达的嵌合抗原受体具有scFv VH-linker-scFv VL-CD8 hinge-CD8TM-4-IBB-CD3ζ的氨基酸结构，所述结构中各部分结构氨基酸序列如表2-1所示。
编号为359的抗DLL3抗体scFv VH及VL、以及LCDR1-3、HCDR1-3的氨基酸序列具体序列见实施例1中的表1-4。
通过基因合成技术制备靶向DLL3嵌合抗原受体基因片段，其氨基酸序列如SEQ ID NO:32所示，具体序列见表2-2。
表2-1.CAR部分元件氨基酸序列
表2-2.DLL3 CAR氨基酸序列
2.2抗PD-L1融合蛋白(aPDL1)、细胞膜型IL7融合蛋白mIL7、aPDL1-mIL7融合蛋白基因片段制备
aPDL1融合蛋白基因片段包含如下基因：kappa前导信号肽、抗PD-L1抗体scfv、连接肽1、人IgG CH2CH3片段。其中接连接肽1的氨基酸序列为G4S，即GGGGS，kappa前导信号肽氨基酸序列为SEQ ID NO:33，抗PD-L1抗体scfv氨基酸序列为SEQ ID NO:34，人IgG CH2CH3片段氨基酸序列为SEQ ID NO:35。
mIL7融合蛋白基因片段包含如下基因：人IL-7细胞因子片段、连接肽2、CD8跨膜区。其中连接肽2氨基酸序列为Ser-Gly Linker，即SGGGSGGGGSGGGGSGGGGSGGGSLQ，人IL-7细胞因子片段氨基酸序列为SEQ ID NO:36，CD8跨膜区氨基酸序列为SEQ ID NO:37。
aPDL1-mIL7融合蛋白基因片段包含aPDL1和mIL7融合蛋白基因片段，两者以2A肽连接，其2A肽采用(GSG)T2A，氨基酸序列为(GSG)EGRGSLLTCGDVEENPGP(SEQ ID NO:38)。SEQ ID NO:33-37具体序列见表2-3。
上述aPDL1、mIL7、aPDL1-mIL7融合蛋白基因由南京金斯瑞生物科技有限公司通过基因合成技术合成，其氨基酸序列如SEQ ID NO:21-23所示，具体序列见表2-4。
表2-3.CAR部分元件氨基酸序列
表2-4.增强功能融合蛋白氨基酸序列

2.3基于piggybac(PB)转座子系统的DLL3 CAR-aPDL1-mIL7载体构建
(1)载体构建方法：
以pBluescript II SK(+)(委托通用生物合成)为起始骨架，在MCS(多克隆位点)区域，插入3'ITR序列，卡那抗性基因序列和5'ITR序列，在3'ITR和5'ITR内侧，插入2xcHS4基因组绝缘序列，在2xcHS4序列内侧，插入EF1a启动子和polyA序列,在EF1a启动子的下游，接入CD8α-DLL3 antibody ScFv HV-linker-ScFv VL-CD8 hinge-CD8TM-4-1BB-CD3ζ(DLL3 CAR)基因序列，以上序列形成Piggybac DLL3 CAR质粒结构(质粒简称DLL3 CAR)。此部分构建使用的具体序列见表2-6。
同时在Piggybac DLL3 CAR质粒结构上通过同源重组的方法将上述2.2部分制备的抗PD-L1融合蛋白(aPDL1)序列或细胞膜型IL7融合蛋白(mIL7)序列或aPDL1-mIL7融合蛋白序列接入DLL3 CAR序列后面，以2A肽连接，其2A肽采用(GSG)P2A，氨基酸序列为(GSG)ATNFSLLKQAGDVEENPGP(SEQ ID NO:42)，形成DLL3 CAR.aPDL1、DLL3 CAR.mIL7、DLL3 CAR.aPDL1.mIL7基因片段结构，其氨基酸序列为SEQ ID NO:43-45，具体序列见表2-5，结构示意图如图4A所示。
表2-5.增强型CAR结构融合蛋白氨基酸序列

表2-6.载体构建所需具体序列

以上构建的载体分别形成三种质粒载体：Piggybac DLL3 CAR-aPDL1质粒载体、Piggybac DLL3 CAR-mIL7质粒载体、Piggybac DLL3 CAR-aPDL1-mIL7质粒载体(分别简称DLL3 CAR.aPDL1、DLL3 CAR.mIL7、DLL3 CAR.aPDL1.mIL7)。图4B示出了piggybac(PB)转座子系统下DLL3 CAR质粒结构示意图，图4C示出了piggybac(PB)转座子系统下DLL3 CAR.aPDL1.mIL7质粒结构示意图。
对由构建的Piggybac DLL3 CAR、Piggybac DLL3 CAR.aPDL1、Piggybac DLL3 CAR.mIL7、Piggybac DLL3 CAR.aPDL1.mIL7质粒载体进行抽提(委托南京金斯瑞完成)，得到转染级Piggybac转座子质粒。
实施例3共表达抗PD-L1及IL7融合蛋白的DLL3 CAR-T细胞制备
3.1人T淋巴细胞的筛选
通过CD3MicroBeads human-lyophilized Kit(Miltenyi Biotech)对外周血单核细胞(PBMC)(购子上海澳能生物)进行磁珠标记，阳性分选出高纯度的CD3阳性T淋巴细胞，分选后的CD3阳性T细胞比例在95％以上。纯化后的T细胞，再利用Dynabeads Human T-Activator CD3/CD28(购买自Thermo Fisher，Cat.No.111.32D)进行T淋巴细胞激活及增殖。
3.2基于Piggybac(PB)转座子系统的CAR基因转导
转座子，又名转座因子，是一类在基因组内可以“跳跃、移动”的遗传因子。PiggyBac转座子(简称PB转座子)具有广泛的转座活性，较少地依赖于宿主因子即可实现基因高效转座。基于piggybac(PB)系统的基因转导包含两部分：PB转座酶(DNA或mRNA形式)和携带目的基因质粒(PB质粒)组成。相比于传统CAR基因转导方式，PB系统不使用病毒，就能将表达CAR的转基因运送到T细胞内，且能承载更多遗传物质，同时无慢病毒转导系统的相关风险，安全性更高。
本实施例中，在上述3.1的T细胞刺激活化后72-96小时，采用2.3中得到的PB质粒：DLL3 CAR、DLL3 CAR.aPDL1、DLL3 CAR.mIL7、DLL3 CAR.aPDL1.mIL7PB质粒进行电转，方法如下：
首先将用于电转的细胞进行重悬，使用吸头或移液管吹散细胞团块，对细胞重悬液进行计数，使用5x 106细胞用于单次电转实验。使用DPBS(GIBCO，14190-144)将5x 106细胞稀释至5ml，室温条件下300xg离心10分钟，尽可能吸弃上清，避免接触到细胞沉淀，加入5ml DPBS重悬清洗细胞，室温条件下300xg离心10分钟，尽可能吸弃上清，避免接触到细胞沉淀，加入100μL电转缓冲液Entranster-E(Engreen,98668-20)重悬细胞，将细胞悬液转移到1.5mL离心管中。将表3-1列出的电转体系中的组分加入到离心管中，混匀。
表3-1.每组电转体系
采用瑞士Lonza公司的4D-Nucleofector细胞核转仪进行电转，将细胞/质粒悬液迅速转移至电击杯中，并轻轻磕击电击杯使细胞悬液充分在电击杯中形成平衡液面，使用程序EO115进行电转。电转后，将电击杯小心取出。加入500ul预热T细胞培养基X-VIVO 15(Lonza,04-418Q)并在37℃培养箱中平衡5分钟，使用微孔上样吸头重悬细胞，轻轻吹打2-3次。将细胞转移至加有2ml预热培养基的12孔板中，37℃培养。电转后4-6小时对细胞进行换液有利于增加细胞存活率。小心吸弃上清，加入预热的新鲜培养基。在37℃,5％CO2培养箱中培养48-72h小时直至检测。
3.3 T细胞CAR基因转导后CAR阳性率测试
在电转48-72h后，获取DLL3 CAR、DLL3 CAR.aPDL1、DLL3 CAR.mIL7、DLL3 CAR.aPDL1.mIL7质粒电转后的四种T细胞(以上细胞简称为：DLL3 CAR-T、DLL3 CAR.aPDL1-T、DLL3 CAR.mIL7-T、DLL3 CAR.aPDL1.mIL7-T)以及未进行CAR质粒电转的T细胞对照进行CAR阳性率流式分析，分析方法如下：
采用生物素标记的抗人IgG(Fab)2抗体(购自jackson immuno,货号：109-065-006)或者人DLL3抗原蛋白(购自ACRO，货号：DL3-H82E4)作为CAR结合蛋白，再用亲和素偶联PE荧光染料通过流式细胞技术检测嵌合抗原受体(CAR)表达，以未转导的T淋巴细胞作为阴性对照，表达不同功能结构的T淋巴细胞其CAR阳性率如表3-2所示，结果表明本发明的CAR阳性率范围在20-30％。
实验结果表明：本发明中DLL3 CAR、DLL3 CAR.aPDL1、DLL3 CAR.mIL7、DLL3 CAR.aPDL1.mIL7结构均可用于制备CAR-T细胞，且共表达aPDL1及mIL7融合蛋白结构的DLL3 CAR-T细胞与未过表达其他功能蛋白的DLL3 CAR-T在CAR阳性率相当。图5A-图5E示出了通过抗人IgG(Fab)2抗体检测表达不同功能结构DLL3 CAR-T细胞的CAR阳性流式示意图，其中图5A示出了未转导CAR的T细胞结果，图5B示出了DLL3 CAR-T细胞的结果，图5C示出了DLL3 CAR.aPDL1-T细胞的结果，图5D示出了DLL3 CAR.mIL7-T细胞的结果，图5E示出了DLL3 CAR.aPDL1.mIL7-T细胞的结果。
图6A-图6E示出了通过DLL3抗原检测表达不同功能结构DLL3 CAR-T细胞的CAR阳性流式示意图，其中图6A示出了未转导CAR的T细胞结果，图6B示出了DLL3 CAR-T细胞的结果，图6C示出了DLL3 CAR.aPDL1-T细胞的结果，图6D示出了DLL3 CAR.mIL7-T细胞的结果，图6E示出了DLL3 CAR.aPDL1.mIL7-T细胞的结果。
图7A示出了抗人IgG(Fab)2抗体检测方法检测不同功能结构DLL3 CAR-T细胞的CAR阳性率结果。图7B示出了人DLL3抗原蛋白检测方法检测不同功能结构DLL3 CAR-T细胞的CAR阳性率结果。
表3-2.表达不同功能结构DLL3 CAR-T细胞的CAR阳性率
3.4不同结构DLL3 CAR-T细胞CAR表达稳定性检测
对DLL3 CAR、DLL3 CAR.aPDL1、DLL3 CAR.mIL7、DLL3 CAR.aPDL1.mIL7质粒电转后的T细胞以及未进行CAR质粒电转的T细胞进行持续培养，同时在电转后第5、7、9、11天进行CAR阳性率检测，分别采用生物素标记的抗人IgG(Fab)2抗体及人DLL3抗原蛋白作为CAR结合蛋白，再用亲和素偶联PE荧光染料通过流式细胞技术检测嵌合抗原受体(CAR)表达，以未转导的T淋巴细胞作为阴性对照， 记录不同结构DLL3 CAR-T在培养不同天数下CAR阳性率的变化。
实验结果表明：本发明中，对比未过表达其他功能蛋白的DLL3 CAR-T，共表达aPDL1及mIL7融合蛋白结构对DLL3 CAR的稳定性未产生影响。
图8A示出了抗人IgG(Fab)2抗体检测方法检测不同功能结构DLL3 CAR-T细胞在不同培养天数下CAR阳性率的变化结果；图8B示出了人DLL3抗原蛋白检测方法检测不同功能结构DLL3 CAR-T细胞在不同培养天数下CAR阳性率的变化结果。
实施例4共表达抗PD-L1及IL7融合蛋白的DLL3 CAR-T细胞表面IL7融合蛋白(mIL7)检测
对DLL3 CAR-T、DLL3 CAR.aPDL1-T、DLL3 CAR.mIL7-T、DLL3 CAR.aPDL1.mIL7-T以及未进行CAR质粒电转的T细胞对照进行细胞膜表面IL7融合蛋白进行流式分析，方法如下：
采用生物素标记的抗人IL7抗体作为人IL7蛋白的结合抗体，再用亲和素偶联PE荧光染料，通过流式细胞技术检测T细胞膜表面IL7蛋白的阳性率，表达不同功能结构的T淋巴细胞其细胞膜上IL7蛋白阳性率如表4-1所示，实验结果表明：本发明中共表达IL7融合蛋白的DLL3 CAR.mIL7-T、DLL3 CAR.aPDL1.mIL7-T细胞能够在T细胞表面检测到人IL7蛋白，且表达效率和CAR阳性率相当，而未共表达IL7融合蛋白的DLL3 CAR-T、DLL3 CAR.aPDL1-T及未进行CAR质粒电转的T细胞的细胞膜表面未检测到IL7蛋白。
图9A-图9E示出了通过抗人IL7抗体检测表达不同结构DLL3 CAR的T淋巴细胞的细胞膜表面IL7蛋白表达的流式示意图，其中图9A示出了未转导CAR的T细胞结果，图9B示出了DLL3 CAR-T细胞的结果，图9C示出了DLL3 CAR.aPDL1-T细胞的结果，图9D示出了DLL3 CAR.mIL7-T细胞的结果，图9E示出了DLL3 CAR.aPDL1.mIL7-T细胞的结果。
表4-1.表达不同功能结构DLL3 CAR-T细胞的细胞膜IL7蛋白检测阳性率
实施例5共表达抗PD-L1及IL7融合蛋白的DLL3 CAR-T细胞上清aPDL1检测
5.1共表达抗PD-L1及IL7融合蛋白的DLL3 CAR-T细胞上清aPDL1蛋白对靶细胞结合实验
本实施例中，对DLL3 CAR-T、DLL3 CAR.aPDL1-T、DLL3 CAR.mIL7-T、DLL3 CAR.aPDL1.mIL7-T以及未进行CAR质粒电转的T细胞按照5×105CAR/T细胞/mL细胞密度培养72小时，收集细胞上清，以小细胞肺癌细胞株SHP-77-hDLL3及SHP-77-hDLL3-hPDL1细胞作为细胞上清中aPDL1融合蛋白结合测试的靶细胞，其中SHP-77-hDLL3细胞株是通过人DLL3蛋白过表达慢病毒(购于吉满生物)感染、筛选获得的高表达人DLL3蛋白的SHP-77细胞，而SHP-77-hDLL3-hPDL1细胞是在SHP-77-hDLL3-hPDL1细胞上构建、筛选获得的人PD-L1过表达细胞株。将收集的上清与SHP-77-hDLL3及SHP-77-hDLL3-hPDL1细胞共孵育，再用抗人IgG Fc偶联APC荧光染料，通过流式细胞技术检测肿瘤细胞表面PD-L1蛋白的阳性率。
实验结果表明：本发明中DLL3 CAR-T、DLL3 CAR.mIL7-T以及未进行CAR质粒电转的T细胞上清中的蛋白均不能对SHP-77-hDLL3及SHP-77-hDLL3-hPDL1细胞产生特异性结合，而共表达aPDL1融合蛋白的DLL3 CAR.aPDL1-T、DLL3 CAR.aPDL1.mIL7-T细胞上清中的蛋白能够对SHP-77-hDLL3-hPDL1细胞产生结合反应，对SHP-77-hDLL3细胞不产生结合反应，说明DLL3 CAR.aPDL1-T、DLL3 CAR.aPDL1.mIL7-T细胞上清中含有aPDL1融合蛋白，且能与PD-L1蛋白结合。
图10A-图10J示出了表达不同功能结构DLL3 CAR-T细胞培养上清对不同肿瘤细胞结合反应的流式示意图。其中图10A-10E示出了表达不同功能结构DLL3 CAR-T细胞培养上清对SHP-77-hDLL3细胞结合反应的流式示意图，图10F-10J示出了表达不同功能结构DLL3 CAR-T细胞培养上清对SHP-77-hDLL3-hPDL1细胞结合反应的流式示意图。
5.2共表达抗PD-L1融合及IL7融合蛋白的DLL3 CAR-T细胞上清aPDL1蛋白浓度测试实验
本实施例中，对DLL3 CAR-T、DLL3 CAR.aPDL1-T、DLL3 CAR.mIL7-T、DLL3 CAR.aPDL1.mIL7-T以及未进行CAR质粒电转的T细胞按照5×105CAR/T细胞/mL细胞密度培养72小时，收集细胞上清，设置3重复，采用抗人IgG酶免试剂盒检测(采购于Novusbio)上清中人IgG蛋白的浓度，以人IgG蛋白的浓度评估aPDL1的分泌含量，表达不同功能结构DLL3 CAR的T淋巴细胞上清中aPDL1的浓度如表表5-1所示，实验结果表明：本发明中，共表达aPDL1融合蛋白的DLL3 CAR.aPDL1-T、DLL3 CAR.aPDL1.mIL7-T细胞上清中能够检测出人IgG蛋白，浓度在20-30ng/mL，而DLL3 CAR-T、DLL3 CAR.mIL7-T以及未进行CAR质粒电转的T细胞上清中未检测出人IgG蛋白，从而说明共表达aPDL1融合蛋白的DLL3 CAR.aPDL1-T、DLL3 CAR.aPDL1.mIL7-T细胞能够正常分泌aPDL1，并释放到细胞上清中。图11示出了表达不同功能结构DLL3 CAR-T细胞培养上清中人IgG蛋白(aPDL1)浓度测试结果。
表5-1.表达不同功能结构DLL3 CAR的T淋巴细胞上清人IgG蛋白(aPDL1)浓度测试结果
实施例6无添加细胞因子条件培养表达不同功能结构DLL3 CAR-T细胞
本实施例中，DLL3 CAR-T、DLL3 CAR.aPDL1-T、DLL3 CAR.mIL7-T、DLL3 CAR.aPDL1.mIL7-T以及未进行CAR质粒电转的T细胞培养至第10天(以T细胞分选为第0天)，通过离心去除培养基中能促进T细胞扩增的细胞因子人IL7和IL15成分，采用同一细胞密度重新接种至新的无添加细胞因子人IL7和IL15培养基中，通过每2-3天的细胞计数，观察细胞的活率、扩增情况，同时在实验终点通过流式细胞技术检测以上细胞的CAR阳性率，实验设置2复孔。表达不同功能结构DLL3 CAR的T淋巴细胞在无因子条件培养下不同天数细胞活率如表6-1所示，表达不同功能结构DLL3 CAR的T淋巴细胞在无因子条件培养下不同天数活细胞数量如表6-2所示，表达不同功能结构DLL3 CAR的T淋巴细胞在无因子条件培养下不同天数CAR阳性率如表6-3所示。
实验结果表明：未共表达mIL7融合蛋白的DLL3 CAR-T、DLL3 CAR.aPDL1-T及未进行CAR质粒电转的T细胞在无因子条件培养下细胞逐渐死亡，其活率及活细胞数量快速下降。而本发明中本共表达IL7融合蛋白的DLL3 CAR.mIL7-T、DLL3 CAR.aPDL1.mIL7-T细胞在无因子条件培养下仍维持较长时间，其活率及活细胞数量缓慢下降，同时通过检测第10天和第19天的CAR阳性率，发现相比未共表达mIL7融合蛋白的DLL3 CAR-T、DLL3 CAR.aPDL1-T细胞，共表达mIL7融合蛋白的DLL3 CAR.mIL7-T、DLL3 CAR.aPDL1.mIL7-T细胞的CAR阳性细胞比例上升更快。图12示出了表达不同功能结构DLL3 CAR-T细胞在无细胞因子条件培养下不同天数细胞活率变化结果，图13示出了表达不同功能结构DLL3 CAR-T细胞在无细胞因子条件培养下不同天数活细胞数量变化结果，图14示出了表达不同功能结构DLL3 CAR的T淋巴细胞在无因子条件培养下不同天数CAR阳性率变化结果。
表6-1.表达不同功能结构DLL3 CAR的T淋巴细胞在无细胞因子条件培养下不同天数细胞活率

表6-2.表达不同功能结构DLL3 CAR的T淋巴细胞在有细胞因子条件培养下不同天数活细胞数量
表6-3.不同功能结构DLL3 CAR的T淋巴细胞在无因子条件培养下不同天数CAR阳性率

基于上述实验，说明共表达mIL7融合蛋白能够增强CAR-T细胞的活力及持续性，同时CAR阳性率的变化说明mIL7融合蛋白主要作用于T细胞自身CAR阳性细胞群体，引发自身CAR阳性细胞群体扩增和保持稳定。
实施例7表达不同功能结构的DLL3 CAR-T细胞体外细胞毒性测试。
7.1表达不同功能结构的DLL3 CAR-T细胞体外细胞杀伤测试
通过模拟产品的作用机制(MOA)建立体外药效试验。以DLL3靶点相关的肺癌细胞株NCI-H460(非小细胞肺癌)、SHP-77(小细胞肺癌)、SHP-77-hDLL3(小细胞肺癌)、SHP-77-hDLL3-hPDL1(小细胞肺癌)作为DLL3 CAR-T细胞功能验证的靶细胞，其中NCI-H460细胞株人DLL3抗原呈低表达，SHP-77细胞株人DLL3抗原呈中表达，SHP-77-hDLL3及SHP-77-hDLL3-hPDL1细胞株人DLL3抗原呈高表达。以上述制备的DLL3 CAR-T、DLL3 CAR.aPDL1-T、DLL3 CAR.mIL7-T、DLL3 CAR.aPDL1.mIL7-T以及未进行CAR质粒电转的T细胞为效应细胞。按照设置3：1、1:1、0.3:1的效靶比(效应细胞：靶细胞)比，建立CAR-T细胞与靶向肿瘤细胞的共培养体系，通过萤光素酶活性法(试剂盒采购于碧云天，货号RG028)检测肿瘤细胞杀伤率来评估CAR-T生物学效力，同时设立未经转导的T细胞与肿瘤细胞共培养对照体系,实验设置2复孔。
图15A-图15D示出了表达不同功能结构的DLL3 CAR-T细胞对肺癌细胞株的体外特异性杀伤结果，图15A示出了表达不同功能结构的DLL3 CAR-T细胞对NCI-H460细胞株的体外特异性杀伤结果，图15B示出了表达不同功能结构的DLL3 CAR-T细胞对SHP-77细胞株的体外特异性杀伤结果，图15C示出了表达不同功能结构的DLL3 CAR-T细胞对人DLL3阳性SHP-77-hDLL3细胞株的体外特异性杀伤结果，图15D示出了表达不同功能结构的DLL3 CAR-T细胞对人DLL3阳性、人PD-L1阳性的SHP-77-hDLL3-hPDL1细胞株的体外特异性杀伤结果。图15A-图15D中T代表未转导CAR的T细胞实验结果。
实验结果所示，在肿瘤细胞数目固定的情况下，表达不同功能结构的DLL3 CAR-T细胞对SHP-77(小细胞肺癌)、SHP-77-hDLL3(小细胞肺癌)、SHP-77-hDLL3-hPDL1(小细胞肺癌)产生特异性杀伤，且与效靶比呈正相关，在效靶比为3:1的条件下，24小时后的杀伤效率在20-50％，而对人DLL抗原低表达的细胞株NCI-H460无显著杀伤效果。同时表达不同功能结构的DLL3 CAR-T细胞在杀伤效率上无显著性差异。该结果说明表达不同功能结构的DLL3 CAR-T细胞能够特异性的杀伤人DLL3抗原表达的肿瘤细胞，且共表达aPDL1融合蛋白和mIL7蛋白未影响DLL3 CAR-T细胞杀伤效果。表7-1给出了表达不同功能结构的DLL3 CAR-T细胞对上述四种肺癌细胞株的杀伤效果。
其中，特异性杀伤检测方法：采用萤火虫萤光素酶报告基因检测试剂盒(采购于碧云天)进行，萤火虫萤光素酶是一种分子量约为61kD的蛋白，在ATP、镁离子和氧气存在的条件下，可以催化luciferin氧化成oxyluciferin。在luciferin氧化的过程中，会发出生物萤光。生物萤光可以通过化学发光仪(luminometer)或液闪测定仪进行测定。它的原理是将萤火素酶基因构建成报告基因(reporter gene)质粒。然后转染细胞，采用CAR-T细胞或者适当药物等处理细胞后，细胞凋亡或坏死而造成的细胞膜结构的破坏会导致细胞浆内的酶释放到培养液里，通过检测从质膜破裂的细胞中释放到培养液中的萤光素酶的活性，就可以实现对细胞毒性的定量分析。
表7-1表达不同功能结构的DLL3 CAR-T细胞对上述四种肺癌细胞株的杀伤效果




7.2表达不同功能结构的DLL3 CAR-T细胞杀伤肿瘤细胞IFN-γ因子释放
通过上述表达不同功能结构的DLL3 CAR-T细胞与四种肿瘤细胞共培养，24小时后收获共培养上清，通过人IFN-γELISA检测试剂盒(采购于Novusbio)检测分泌至培养基上清中细胞因子(INF-γ)含量来评估CAR-T生物学效力，实验结果表明：表达不同功能结构的DLL3 CAR-T杀伤DLL3抗原阳性肿瘤细胞后细胞因子IFN-γ释放水平高于阴性对照(未转导CAR的T细胞)至少2倍以上，且对比DLL3 CAR-T，共表达aPDL1融合蛋白和mIL7蛋白未影响DLL3 CAR-T细胞IFN-γ因子释放，表7-2给出了未转导CAR的T细胞，本发明的表达不同功能结构的DLL3 CAR-T细胞对NCI-H460、SHP-77、SHP-77-hDLL3、SHP-77-hDLL3-hPDL1四种肺癌细胞株杀伤后IFN-γ释放水平。图16A-图16D示出了本发明的表达不同功能结构的DLL3 CAR-T细胞对四种肺癌细胞杀伤后IFN-γ释放结果，其中图16A示出了表达不同功能结构的DLL3 CAR-T与NCI-H460细胞株共培养后的IFN-γ释放，图16B示出了表达不同功能结构的DLL3 CAR-T与SHP-77细胞株共培养后的IFN-γ释放，图16C示出了表达不同功能结构的DLL3 CAR-T与SHP-77-hDLL3细胞株共培养后的IFN-γ释放，图16D示出了表达不同功能结构的DLL3 CAR-T与SHP-77-hDLL3-hPDL1细胞株共培养后的IFN-γ释放。图16A-16D中T代表未转导CAR的T细胞的IFN-γ释放。
其中，细胞因子检测方法：采用人IFN-γELISA检测试剂盒(采购于Novusbio)进行，是基于抗原或抗体的固相化及抗原或抗体的酶标记。结合在固相载体表面的抗原或抗体仍保持其免疫学活性，酶标记的抗原或抗体既保留其免疫学活性，又保留酶的活性。进行检测时，样品中的受检物质(抗原或抗体)与固定的抗体或抗原结合。通过洗板除去非结合物，再加入酶标记的抗原或抗体，此时，能固定下来的酶量与样品中被检物质的量相关。通过加入与酶反应的底物后显色，根据颜色的深浅可以判断样品中物质的含量，进行定性或定量的分析。
表7-2表达不同功能结构的DLL3 CAR-T细胞对四种肺癌细胞株杀伤后IFN-γ释放结果


基于上述实验，说明共表达aPDL1融合蛋白及mIL7融合蛋白并未影响(降低或失效)DLL3 CAR结构对DLL3抗原阳性肿瘤细胞的杀伤能力，但在24小时杀伤时间内也并未见共表达aPDL1融合蛋白及mIL7融合蛋白对杀伤效率有提升效果。主要原因在于CAR-T细胞对肿瘤细胞时间短杀伤，aPDL1融合蛋白及mIL7融合蛋白的功能很难体现。
实施例8表达不同功能结构的DLL3 CAR-T细胞体外持续杀伤肿瘤能力测试
为了评估共表达aPDL1融合蛋白及mIL7融合蛋白的DLL3 CAR-T细胞在体外长期抗肿瘤的增强作用，本发明设计了本实施例7。通过上述制备DLL3 CAR-T、DLL3 CAR.aPDL1-T、DLL3 CAR.mIL7-T、DLL3 CAR.aPDL1.mIL7-T以及未进行CAR质粒电转的T细胞作为效应细胞，以人小细胞肺癌SHP-77细胞株为靶细胞，按照1:10的效靶比(效应细胞：靶细胞)比，采用无细胞因子共培养体系，每间隔3天添加一次肿瘤细胞重复刺激，通过检测CAR-T细胞和肿瘤细胞在共培养体系中的数量变化来评估表达不同功能结构的DLL3 CAR-T细胞持续杀瘤能力。图17A-图17B示出了表达不同功能结构的DLL3 CAR-T对DLL3表达阳性细胞SHP-77长期抗肿瘤作用结果，其中图17A示出了表达不同功能结构的DLL3 CAR-T细胞对DLL3表达阳性细胞SHP-77长期抗肿瘤作用的肿瘤细胞数量变化结果，图17B示出了表达不同功能结构的DLL3 CAR-T对DLL3表达阳性细胞SHP-77长期抗肿瘤作用的CAR阳性T细胞数量变化结果。
实验结果表明，在效应细胞：靶细胞在1：10的情况下，通过5轮肿瘤细胞刺激，共表达mIL7融合蛋白的DLL3 CAR.mIL7-T、DLL3 CAR.aPDL1.mIL7-T细胞对肿瘤细胞的清除作用明显优于未共表达IL7融合蛋白的DLL3 CAR-T、DLL3 CAR.aPDL1-T细胞，同时未进行CAR质粒电转的T细胞对肿瘤细胞 没有清除作用。而且共表达mIL7融合蛋白的DLL3 CAR.mIL7-T、DLL3 CAR.aPDL1.mIL7-T细胞在持续性杀伤中保持良好的细胞扩增，其细胞持续性优于未共表达IL7融合蛋白的DLL3 CAR-T、DLL3 CAR.aPDL1-T细胞。
炎症性肿瘤微环境提供了调节PD-L1表达的各种因子。IFN-γ主要由效应T细胞和NK细胞分泌，是各种肿瘤细胞PD-L1最有效的诱导剂。在机制上，IFN-γ诱导的PD-L1上调是通过JAK1/2–STAT1激活介导的，最终由IFN-γ调节因子1(IRF1)与PD-L1启动子直接结合(BMB Rep.2021Aug 31；54(8):403–412)。而CAR-T细胞在杀伤肿瘤细胞过程中会释放细胞因子IFN-γ，从而诱发肿瘤细胞PD-L1上调，进一步与CAR-T细胞的PD-1结合，造成CAR-T细胞衰竭。
本发明中共表达aPDL1融合蛋白的DLL3 CAR-T细胞能够分泌aPDL1融合蛋白，能够对肿瘤细胞的PD-L1蛋白进行结合，因此基于实施例7，对CAR-T细胞杀伤后的SHP77肿瘤细胞的PD-L1蛋白进行流式分析，实验结果显示，表达不同功能结构的DLL3 CAR-T细胞在杀伤SHP-77细胞时，SHP-77细胞的细胞膜表明PD-L1蛋白表达明显上调，而共表达aPDL1融合蛋白的DLL3 CAR.aPDL1-T、DLL3 CAR.aPDL1.mIL7-T细胞分泌的aPDL1融合蛋白能够结合SHP-77细胞的PD-L1蛋白，降低PD-L1蛋白的检测值，从而阻断PD-1与PD-L1通路。表8-1给出了未转导CAR的T细胞及本发明的表达不同功能结构的DLL3 CAR-T细胞杀伤SHP-77肿瘤细胞后剩余肿瘤细胞PD-L1的表达结果。图18示出了SHP-77细胞在表达不同功能结构的DLL3 CAR-T细胞持续杀伤后PD-L1蛋白表达结果。
表8-1 SHP-77细胞在表达不同功能结构的DLL3 CAR-T细胞持续杀伤后PD-L1蛋白表达结果
基于上述实验，说明本发明中共表达aPDL1融合蛋白的DLL3 CAR-T细胞分泌的aPDL1融合蛋白能够结合肿瘤细胞的PD-L1蛋白，从而阻断PD-1与PD-L1通路。而共表达mIL7融合蛋白的DLL3 CAR-T细胞能够增强CAR-T细胞的扩增能力及持续性。共表达aPDL1融合蛋白及mIL7融合蛋白的DLL3 CAR-T细胞保留了以上两种结构的功能，增强了DLL3 CAR-T细胞持续杀伤肿瘤的能力。
实施例9动物药效实验
在本实施例7中，建立人小细胞肺癌肿瘤细胞负荷的免疫缺陷型小鼠药效模型，用于评估共表达aPDL1融合蛋白及mIL7融合蛋白的DLL3 CAR-T细胞细胞在小鼠体内的药效。基于体外研究以雌性NCG小鼠(购自集萃药康)经背皮注射5×106个人小细胞肺癌细胞SHP-77，接种SHP-77细胞第6天(肿瘤体积在50-60mm3大小)给药，共设置3组，分别为T细胞(未转导CAR的T细胞)组、未共表达aPDL1融合蛋白及mIL7融合蛋白的DLL3 CAR-T细胞(DLL3 CAR-T)组和共表达aPDL1融合蛋白及mIL7融合蛋白的DLL3 CAR-T细胞(DLL3 CAR.aPDL1.mIL7-T)组，其中T细胞组按照5×106T细胞数量/小鼠剂量给药，DLL3 CAR-T细胞组按照5×106CAR-T细胞数量/小鼠剂量给药，DLL3 CAR.aPDL1.mIL7-T细胞组按照2×106CAR-T细胞数量/小鼠剂量给药，所有条件组动物数量均为5只。给药后每周2次量瘤；绘制肿瘤生长曲线。图19示出了DLL3 CAR-T细胞、DLL3 CAR.aPDL1.mIL7-T细胞及未转导CAR的T细胞给药后小细胞肺癌SHP-77肿瘤荷瘤NCG小鼠的肿瘤大小变化结果。实验结果表明：给药后32天,DLL3 CAR-T细胞、DLL3 CAR.aPDL1.mIL7-T细胞均对小鼠体内SHP-77肿瘤有抑制作用，而低剂量下(2×106CAR-T细胞数量/小鼠)DLL3 CAR.aPDL1.mIL7-T细胞对肿瘤的抑制作用显著优于高剂量下(5×106CAR-T细胞数量/小鼠)DLL3 CAR-T细胞，说明共表达aPDL1融合蛋白及mIL7融合蛋白能够显著增强DLL3 CAR-T细胞的抗肿瘤能力。

Claims (15)

  1. 一种抗DLL3抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含下述3个轻链互补决定区和3个重链互补决定区，所述抗体或其抗原结合片段的3个轻链互补决定区包含如SEQ ID NO:22所示LCDR1,如SEQ ID NO:23所示LCDR2,如SEQ ID NO:24所示LCDR3；和3个重链互补决定区包含如SEQ ID NO:18所示HCDR1，如SEQ ID NO:19所示的HCDR2,如SEQ ID NO:20所示的HCDR3。
  2. 根据权利要求1所述抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含与SEQ ID NO:21所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的轻链可变区(VL)，和与SEQ ID NO:17所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的重链可变区(VH)；
    优选的，所述抗体包含与SEQ ID NO:25所示序列具有至少95％、96％、97％、98％、99％或100％同一性的重链恒定区，和包含与SEQ ID NO:26所示序列具有至少95％、96％、97％、98％、99％或100％同一性的轻链恒定区。
  3. 一种靶向DLL3抗原的嵌合抗原受体(CAR)，其特征在于，所述CAR包含DLL3抗原结合结构域、跨膜域和胞内信号转导结构域，其中所述DLL3抗原结合结构域是scFv,其中所述scFv为权利要求1-2任一项所述抗体或其抗原结合片段。
  4. 根据权利要求3的靶向DLL3抗原的嵌合抗原受体，其特征在于，所述CAR还包含铰链区、信号肽和共刺激信号域中的一种或多种；
    优选地，所述跨膜结构域为CD8跨膜区，所述铰链区为CD8铰链区，所述细胞内信号传导结构域为CD3ζ胞内信号域，所述信号肽为CD8α信号肽，或所述共刺激信号域为4-1BB或者CD28共刺激信号域。
  5. 根据权利要求3或4的靶向DLL3抗原的嵌合抗原受体，其特征在于，所述CAR从N端到C端依次包含CD8α信号肽、DLL3抗体scFv VH-linker-DLL3抗体scFv VL、CD8铰链区、CD8跨膜区、4-1BB共刺激信号和CD3ζ胞内信号域；
    优选的，所述CD8α信号肽包含与SEQ ID NO:27所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；所述CD8铰链区及跨膜区包含与SEQ ID NO:28所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；所述4-1BB共刺激信号域包含与SEQ ID NO:29所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；或者所述CD3ζ胞内信号域包含与SEQ ID NO:30所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；
    更优选的，所述CAR的氨基酸序列如SEQ ID NO:32所示。
  6. 根据权利要求3-5任一所述的靶向DLL3抗原的嵌合抗原受体，其特征在于，所述CAR包含如下元件i)和/或ii)：
    i)免疫抑制分子表达元件；
    ii)细胞膜型白介素和/或分泌型趋化因子的表达元件，
    优选的，所述免疫抑制分子选自：PD1和PDL1中的一个或者多个；所述细胞膜型白介素选自细胞膜型IL2细胞因子、细胞膜型IL4细胞因子、细胞膜型IL7细胞因子、细胞膜型IL9细胞因子、细胞膜型IL10细胞因子、细胞膜型IL15细胞因子、、细胞膜型IL18细胞因子、细胞膜型IL21细胞因子、细胞膜型IL23细胞因子、细胞膜型IL24细胞因子、细胞 膜型IL36细胞因子中的一个或者多个；所述分泌型趋化因子选自分泌型CCL1趋化因子、分泌型CCL2趋化因子、分泌型CCL3趋化因子、分泌型CCL5趋化因子、分泌型CCL7趋化因子、分泌型CCL15趋化因子、分泌型CCL16趋化因子、分泌型CCL19趋化因子、分泌型CCL20趋化因子、分泌型CCL21趋化因子、分泌型CXCL4趋化因子、分泌型CXCL9趋化因子、分泌型CXCL10趋化因子、分泌型CXCL11趋化因子、分泌型CXCL1趋化因子中的一个或者多个。
  7. 根据权利要求6所述的靶向DLL3抗原的嵌合抗原受体，其特征在于，所述CAR包含抗PDL1蛋白表达元件和/或细胞膜型IL7细胞因子表达元件；
    优选的，所述抗PDL1表达元件依次包含kappa前导信号肽、抗PD-L1抗体scfv、连接肽1和人IgG CH2CH3片段；所述细胞膜型IL7细胞因子表达元件依次包含人IL-7细胞因子片段、连接肽2、CD8跨膜区；
    优选的，所述抗PDL1表达元件中的kappa前导信号肽包含与SEQ ID NO:33所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；所述抗PDL1抗体scfv包含与SEQ ID NO:34所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；连接肽1的氨基酸序列为GGGS；人IgG CH2CH3片段包含SEQ ID NO:35所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；所述细胞膜型IL7细胞因子表达元件中的人IL-7细胞因子片段包含与SEQ ID NO:36所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；连接肽2氨基酸序列为SGGGSGGGGSGGGGSGGGGSGGGSLQ；CD8跨膜区氨基酸序列包含与SEQ ID NO:37所示氨基酸序列具有至少95％、96％、97％、98％、99％或100％同一性的氨基酸序列；所述PDL1表达元件和细胞膜型IL7细胞因子表达元件两者以2A肽连接，所述2A肽序列如SEQ ID NO:38所示；
    优选的，所述CAR的氨基酸序列如SEQ ID NO:43、SEQ ID NO:44或SEQ ID NO:45所示。
  8. 一种核酸，其编码权利要求1-2任一所述的抗DLL3抗体或其抗原结合片段或编码权利要求3-7任一项所述的靶向DLL3抗原的嵌合抗原受体。
  9. 一种表达盒，其包含权利要求8所述的核酸。
  10. 一种载体，其包含权利要求8所述核酸或权利要求9所述表达盒。
  11. 一种细胞，其包含权利要求8所述核酸或权利要求9所述表达盒或权利要求10所述载体；优选地，所述细胞为工程化的免疫细胞；更优选地，所述工程化的免疫细胞为T细胞；更优选地，所述T细胞为原代来源T细胞或者iPSC分化来的T细胞，所述iPSC分化来的T细胞为γδT细胞、DNT细胞或NKT细胞。
  12. 一种药物组合物，其特征在于，所述药物组合物含有权利要求1-2任一所述的抗DLL3抗体或其抗原结合片段、权利要求3-7任一项所述的靶向DLL3抗原的嵌合抗原受体、权利要求8所述核酸、权利要求9所述表达盒、权利要求10所述载体、或权利要求11所述细胞，及其药物上可接受的载体。
  13. 一种试剂盒，其特征在于，所试剂盒含有权利要求1-2任一所述的抗DLL3抗体或其抗原结合片段、权利要求3-7任一项所述的靶向DLL3抗原的嵌合抗原受体、权利要求8所述 核酸、权利要求9所述表达盒、权利要求10所述载体、或权利要求11所述细胞，及试剂盒所需的缓冲溶液。
  14. 权利要求1-2任一所述的抗DLL3抗体或其抗原结合片段、权利要求3-7任一项所述的靶向DLL3抗原的嵌合抗原受体、或权利要求8所述核酸、权利要求9所述表达盒、权利要求10所述载体、权利要求11所述细胞、权利要求12所述药物组合物、或权利要求13所述试剂盒用于预防、治疗、检测或诊断与DLL3相关的疾病的应用；优选地，所述与DLL3相关的疾病是DLL3高表达疾病；更优选地，所述疾病是DLL3高表达癌症或肿瘤；更优选地，所述癌症或肿瘤选自肺癌、黑色素瘤、甲状腺髓样癌、胶质母细胞瘤、前列腺癌、神经内分泌癌中的一种或多种；更优选地，所述癌症是肺癌，特别是小细胞肺癌。
  15. 一种制备工程化免疫细胞的方法，其特征在于，包括以下步骤：
    (1)提供一待改造的免疫细胞；和
    (2)将权利要求8所述核酸、或权利要求9所述表达盒、或权利要求10所述载体导入到所述免疫细胞；
    优选地，所述免疫细胞为T细胞；更优选地，所述T细胞为原代来源T细胞或者iPSC分化来的T细胞，所述iPSC分化来的T细胞为γδT细胞、DNT细胞或NKT细胞。
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