WO2023015832A1 - 一种细胞激活依赖性分泌系统及应用 - Google Patents

一种细胞激活依赖性分泌系统及应用 Download PDF

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WO2023015832A1
WO2023015832A1 PCT/CN2021/143120 CN2021143120W WO2023015832A1 WO 2023015832 A1 WO2023015832 A1 WO 2023015832A1 CN 2021143120 W CN2021143120 W CN 2021143120W WO 2023015832 A1 WO2023015832 A1 WO 2023015832A1
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cell
cells
activation
car
secretion system
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French (fr)
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谢国柱
刘梓燊
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南方医科大学南方医院
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C12N5/0646—Natural killers cells [NK], NKT cells
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    • C12N2510/00—Genetically modified cells

Definitions

  • the invention relates to the fields of cell biology and immunotherapy, and more specifically, the invention relates to a cell activation-dependent secretion system and its application.
  • CAR Chimeric antigen receptor
  • CAR-T CAR-modified T cells
  • Tumor microenvironment is an immunosuppressive local internal environment composed of tumor cells, stromal cells, immune cells and active mediators secreted by tumor tissue (see Figure 1).
  • TME not only restricts the infiltration of CAR-engineered immune cells (such as CAR-T or CAR-NK cells), but also prevents effector immune cells that have infiltrated into the tumor from functioning effectively.
  • immune checkpoint molecules such as PD-1/PD-L1, NKG2A/HLA-E, TIGIT/CD155
  • soluble immunosuppressive molecules such as TGF- ⁇ , PGE2 expressed by tumor cells or stromal cells in the TME
  • immunosuppressive metabolites such as lactic acid, ADO
  • CAR-modified immune cells such as CAR-T or NK cells, CAR-T/NK
  • immunosuppressive molecule-specific antibodies such as anti-NKG2A antibodies
  • cytokines with anti-tumor activity such as IL-2, IL-15, etc.
  • Another approach is to genetically engineer NK cells to directly express immune checkpoint blocking antibodies or immune-activating cytokines to promote the persistence and anti-tumor activity of CAR-T/NK.
  • NK cells themselves may lead to systemic toxicity, such as autoimmune toxicity associated with immune checkpoint inhibitors, IL-15-related bleeding events and gastric Intestinal ischemic necrosis [5] .
  • CAR-T Chimeric antigen receptor T cell, chimeric antigen receptor T cell
  • T cells are directly combined with specific antigens on the surface of tumor cells to be activated, directly kill tumor cells by releasing perforin, granzyme B, etc., and at the same time recruit human endogenous immune cells to kill tumor cells by releasing cytokines, In this way, the purpose of treating tumors can be achieved, and immune memory T cells can also be formed to obtain a specific long-term anti-tumor mechanism.
  • Granzyme B (Granzyme B, Gran B) is currently found to be the most active granzyme inducing target cell apoptosis in CTLs and NK cells, and plays a central role in mediating target cell apoptosis.
  • Gran B cleaves specific substrates containing Ile/Val, Glu/Met/Gln, Pro/Xaa and Asp amino acid sequences (P4-P1), triggering target cell apoptosis (see Figure 4) [6] .
  • a large amount of GranB secreted around CTLs or NK cells can also effectively cleave molecules containing specific amino acid sequences.
  • the first-generation CAR-T structure only contains the antigen recognition signal and CD3 ⁇ signal activation domain, and has limited proliferation ability in vivo; therefore, the second-generation and third-generation CAR-T have added one and two co-stimulatory molecules, respectively, to enhance T cell proliferation. Proliferative activity, cytotoxicity and survival time in vivo.
  • the fourth-generation CAR-T cell technology also known as TRUCK T cell technology, is through the transfer of cytokines containing nuclear factor of the activated T cell (NFAT) into CAR-T cells and transcription of the corresponding elements.
  • the expression vector can promote the activation of CAR-T cells stimulated by the target antigen to secrete the target cytokines in the local tumor.
  • TCR T cell receptor
  • ITAM immunoreceptor tyrosine-activating motif
  • the fourth-generation CAR-T technology uses this principle to introduce the cytokine expression cassette containing NFAT response elements into CAR-T cells. After the CAR-T cells bind and activate the target cell antigen, they can induce the expression of the target cytokine. Expression and secretion (such as IL-12, see Figure 2) [9] .
  • TURCK CAR-T technology The purpose of TURCK CAR-T technology is to induce the expression and secretion of target cytokines after CAR-T cells bind and activate the target antigen, so as to make the cytokines accumulate in the tumor tissue as much as possible and reduce the systemic toxicity caused by peripheral secretion.
  • this TRUCK T cell technology has great safety risks [8] , mainly due to the "leakage" of NFAT promoter, NFAT activation triggered by T cells' own TCR (endogenous TCR), and CD3 signal-independent NFAT activation (see Figure 6) [9-11] .
  • TILs tumor-infiltrating lymphocytes
  • TRUCK tumor-infiltrating lymphocytes
  • cytokine-inducible expression cassettes must be two different promoters. In order to prevent the inducible expression cassette from being transactivated by the strong constitutive CAR promoter, two viral vector transductions are required.
  • the ideal technology is to enable CAR-T/NK cells to secrete the required effector molecules locally in the tumor tissue, so as to improve the tumor microenvironment, achieve effective immune cell infiltration and efficiently kill tumor cells, and at the same time reduce or avoid Systemic toxicity mediated by secretion of the active molecule into the blood.
  • the purpose of the present invention is to provide a new cell system, which can be used in various aspects such as cell signal transduction research and protein molecular expression research after the binding of various receptor ligands.
  • This system is called cell activation-dependent in the present invention. secretory system.
  • a cell activation-dependent secretion system in one solution of the present invention, includes cells and vectors, and the vectors are introduced into cells to express secretion elements that can be secreted to the cell membrane or around the cell, and the cleavage elements produced after cell activation The molecule cleaves the secretory element.
  • a cell activation-dependent secretion system in one solution of the present invention, includes cells and vectors, and the vectors are introduced into cells to express secretion elements that can be secreted to the cell membrane or around the cell, and the cleavage elements produced after cell activation The molecule cleaves the secretory element.
  • the secretory element is expressed on the same vector as the cell-specific activating receptor, and then the secretory element is secreted to the cell membrane or the cell periphery
  • the secretory element and the cell-specific activating receptor are expressed on the same carrier, and the carrier includes a cell-specific activating receptor composed of a ligand binding domain, a transmembrane domain, and an intracellular domain region, and the secretory element domain.
  • the secretory element and the cell-specific activating receptor are co-expressed on the same vector driven by a single promoter, and the vector includes a ligand binding domain, a transmembrane domain, and an intracellular domain. Consists of a cell-specific activation receptor domain, and a secretory element domain.
  • the secretory element and the cell-specific activating receptor are expressed on the same vector driven by different promoters, and the vector includes a ligand binding domain, a transmembrane domain, an intracellular domain Consists of a cell-specific activation receptor domain, and a secretory element domain.
  • the secretory element domain includes a leader sequence region, a bioactive molecule region, and a cleavage molecule substrate region, and the bioactive molecule region is connected to the cleavage molecule substrate region; the biological activity expressed by the bioactive molecule region Active molecules play the role of biologically active molecules after the cells are activated; the cleavage molecule substrate region expresses the cleavage molecule substrates, and the cleavage molecules produced after the cells are activated cut the cleavage molecule substrates to release the bioactive molecules linked to them .
  • the secretory element domain includes a leader sequence region, a biologically active molecule region, a cleavage molecule substrate region and a transmembrane region, and the bioactive molecule region is connected to the cleavage molecule substrate region; the bioactive molecule
  • the biologically active molecule expressed in the region plays the role of a biologically active molecule after the cell is activated; the cutting molecule substrate region expresses a cutting molecule substrate, and the cutting molecule produced after the cell is activated cuts the cutting molecule substrate and releases the connection with it bioactive molecules.
  • the intracellular domain and the secretory element domain are connected by an element that can express multiple polycistrons on a single vector; preferably, the element is a self-cleaving element expressing a self-cleaving polypeptide or an internal Ribosome entry site (IRES) elements.
  • the element is a self-cleaving element expressing a self-cleaving polypeptide or an internal Ribosome entry site (IRES) elements.
  • the cell-specific activating receptor is a receptor molecule produced by the expression of an endogenous gene or an exogenous gene of the cell, which activates the cell to produce a cleavage molecule after binding to a ligand; preferably, the cell-specific Sexually activating receptors are chimeric antigen receptors (CARs) or T cell antigen receptors (TCRs).
  • CARs chimeric antigen receptors
  • TCRs T cell antigen receptors
  • the cell-specific activating receptor is a chimeric antigen receptor CAR including a CAR for T cells, or a CAR for NK cells; wherein the CAR for NK cells contains scFv, CD8hinge, NKG2D TM, 2B4 IC, CD3 ⁇ structure; CAR for T cells contains scFv, CD8hinge, CD8 TM, 4-1BB IC, CD3 ⁇ structure.
  • the cleavage molecule is an enzyme or a protein with cleavage activity, preferably the cleavage molecule is a cleavage enzyme, more preferably the cleavage molecule is a granzyme, and the granzyme can be granzyme A, granzyme B. Granzyme H, Granzyme K, Granzyme B, Granzyme M.
  • the cleavage enzyme is caspase, preferably caspase-8, caspase-10, caspase-3, caspase-7.
  • the biologically active molecule is one or more of protein molecules and polypeptides;
  • the protein molecules include antibodies, antibody active fragments, cytokines, receptors, ligands, etc.;
  • bioactive molecules include molecules that promote the proliferation or differentiation of activated cells or change the activity of cells, molecules that promote apoptosis or change the activity of target cells, molecules that recruit other cells or can change the local microenvironment .
  • the biologically active molecules are cytokines/chemokines, such as cytokines such as XCL1, IFN ⁇ , IL-12, IL-2, IL-7, IL-15, antibodies or antibody fragments, polypeptide fragments , small molecule inhibitors, etc.
  • cytokines/chemokines such as XCL1, IFN ⁇ , IL-12, IL-2, IL-7, IL-15, antibodies or antibody fragments, polypeptide fragments , small molecule inhibitors, etc.
  • the cleaved molecular substrate is an enzymatic cleavage substrate polypeptide; the intracellular domain and the secretory element domain are connected through the self-cleavage region expressing the self-cleavage polypeptide; the activated cells are immune cells , preferably T cells, NK cells, ⁇ T, NKT, macrophages, dendritic cells or B cells, the enzyme-cleaved substrate polypeptide sequence contains IEFD, IEPD, VGPD or IEAD. In one embodiment, the enzyme cleavage substrate polypeptide sequence contains ELQTDG, DEVDR, RIEADS.
  • the present invention also provides a nucleic acid, the nucleic acid includes a cell-specific activation receptor region and a secretory element domain, the nucleic acid is inserted into an expression vector, and then introduced into a cell, and in the cell The cell-specific activation receptor and secretory element are expressed, the secretory element is secreted and expressed to the cell membrane or the periphery of the cell, and the cleavage molecule produced after cell activation cleaves the secretory element.
  • the present invention also provides an expression vector, which expresses a secretory element that can be secreted to the cell membrane or around the cell after the vector is introduced into the cell, and the cleavage molecule produced after the cell is activated cleaves the secretory element; preferably
  • the carrier includes a cell-specific activation receptor region and a secretory element domain; more preferably, the carrier includes a chimeric antigen receptor region and a secretory element domain.
  • the present invention also provides a host cell containing the aforementioned expression vector.
  • the host cells are immune cells, preferably T cells, NK cells, ⁇ T cells, NKT cells, macrophages, dendritic cells or B cells.
  • the present invention also provides a pharmaceutical composition, which includes any of the aforementioned cell activation-dependent secretion systems, nucleic acids, expression vectors or host cells, and pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention also provides an application of a cell activation-dependent secretion system
  • the system is any one of the above-mentioned systems, and the system is used for preparing cell preparations or cell medicines.
  • the cell preparation or cell drug can be activated by binding to the ligand on the target cell, releasing bioactive molecules, and the bioactive molecule promotes the proliferation or differentiation or activity change of the activated cell, and promotes the apoptosis or activity change of the target cell , or recruit other cells.
  • the cell-specific activating receptor in the system is a chimeric antigen receptor CAR
  • the chimeric antigen receptor CAR expressed on the cell surface in the system binds to the surface antigen of the target cell
  • the The cells are activated to produce cleavage molecules, and the cleavage molecules cleave the secretory elements that are co-expressed with the chimeric antigen receptor CAR on the cell membrane or in the cell periphery, and the bioactive molecules located in the secretory elements are released, and the released bioactive molecules further promote
  • the killing effect of the cells in the system on the target cells may maintain or improve the effector function of the cells in the system and their ability to expand and persist.
  • the cells in the system of the present invention are immune cells, preferably T cells, NK cells, ⁇ T cells, NKT cells, macrophages, dendritic cells or B cells.
  • the preparation/drug described in the present invention is a liquid preparation, and the drug is an injection.
  • the system, host cell, and pharmaceutical composition of the present invention can also be used together with other drugs, preparations, devices, and therapies that have therapeutic and adjuvant treatments to enhance the effect.
  • the target cells of the present invention are tumor cells, and the tumor cells are from solid tumors or humoral tumors, preferably from solid tumors.
  • the solid tumors include malignant tumors of the head and neck, thoracic and mediastinal tumors, digestive system tumors, genitourinary system malignant tumors, bone and soft tissue tumors, breast cancer, lymphoma, skin malignant tumors, melanoma, and central nervous system tumors. one or more.
  • the target cells of the present invention are tumor cells
  • the tumors are pharyngeal cancer, laryngeal cancer, oral cancer, esophageal cancer, thymus cancer, lung cancer, pancreatic cancer, colon cancer, colorectal cancer, gastric cancer , liver cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, testicular cancer, osteosarcoma, leiomyosarcoma, breast cancer, lymphoma, skin malignancies, melanoma, meningioma, colloid one or more of the tumors.
  • the present invention also provides an application of a cell activation-dependent secretion system, the system is any one of the aforementioned systems, and the system can be used to prepare a kit.
  • the present invention also provides a kit, which includes one or more of the aforementioned systems, nucleic acids, vectors or host cells.
  • the kit also includes reagent containers, instructions for use of the kit, and/or packaging materials, etc.
  • the cell-specific activating receptor and the secretory element are not located on the same nucleic acid or carrier, they can be respectively located on different nucleic acids or vectors, and when used, the cell-specific activating receptor region Nucleic acids or vectors for the domain of the secretory element and the secretory element are transduced into the same cell and expressed to the cell membrane or the peripheral region of the cell, respectively. After the cell-specific activating receptors located on the cell membrane are activated by exogenous ligands, the cells produce cleavage molecules that cleave the secretory element to release the biologically active molecules in the secretory element.
  • the cell-specific activation receptor and the secretory element are not located on the same piece of nucleic acid or carrier, they can be respectively located on different nucleic acids or carriers, and when used, contain the Nucleic acids or vectors for cell-specific activating receptor domains and secretory element domains are transduced separately into the same cell. Cell-specific activating receptors expressed in the cell-specific activating receptor region localize to the cell membrane.
  • the receptor can activate the carrier expressing the secretory element in the cell, and the secretory element is secreted to the cell membrane or the periphery of the cell, and then, the cell-specific activating receptor and the external
  • the cleavage molecule produced upon activation of the source ligand binding cleaves the secretory element to release the bioactive molecule in the secretory element.
  • the biologically active molecule can promote the killing effect of cells containing cell-specific activating receptors on target cells with ligands, or maintain or improve the effector function of cells containing cell-specific activating receptors Its ability to expand and persist can improve the tumor microenvironment and induce systemic anti-tumor immune response.
  • the present invention provides a cell activation-dependent secretion system.
  • the system includes secretory elements secreted and expressed to the cell membrane or cell periphery after being introduced into cells, and the cleavage molecules produced after cell activation cleave the secreted elements. element.
  • the cells are immune cells, preferably T cells, NK cells, ⁇ T cells, NKT cells, macrophages, dendritic cells or B cells.
  • the preparation method of cell activation-dependent secretion system comprises:
  • the self-cleaving polypeptide is self-cleaving 2A polypeptide; the cleaving enzyme is granzyme B; and the cleaving enzyme substrate polypeptide sequence contains IEFD, IEPD or VGPD.
  • CAR-T/NK cells bind to the target antigen through the CAR, and the cleavage enzyme secreted to the extracellular space in the final stage of killing the target cell cuts the membrane-bound biologically active molecule containing the cleavage substrate polypeptide , to release bioactive molecules.
  • the self-cleaving polypeptide may be a protein peptide capable of self-cleaving after expression, such as T2A, P2A, E2A, and F2A;
  • the vector is selected from the group consisting of plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, DNA, RNA, transposons, or combinations thereof; preferred vectors of the present invention are lentiviral vector.
  • the cell activation-dependent secretion system developed by the present invention uses CAR-T/NK cells to exert the target cell killing effect after activation and secrete in the final stage
  • the enzymatic cleavage activity of Gran B (that is, at the end stage of the effect) is used to cleave and release bioactive molecules, which avoids unintended secretion caused by non-lethal activation signals.
  • the secreted bioactive molecules are more effective in CAR-
  • the accumulation of T/NK cells killing target cells in tumor tissue is beneficial to play a role in the local tumor microenvironment, thereby avoiding the systemic toxicity mediated by a large number of unintended secretions.
  • NK cells do not have T cell receptors (TCR)
  • the engineered CAR-NK cells constructed by the technical scheme of the present invention avoid the non-tumor tissue induced by the activation of endogenous TCR by other antigens (non-tumor antigens). Endocrine, further improving safety.
  • the activation-dependent secretory CAR-T/NK cells constructed by the present invention are more likely to achieve safe targeted delivery of bioactive molecules to the tumor microenvironment in immunotherapy , to achieve the purpose of reducing the toxic and side effects of secretory active molecules.
  • it is expected to transform CAR-T/NK cells into a safe and efficient "miniature pharmaceutical factory", which can produce the required bioactive molecules in the tumor to overcome the immune suppression mediated by the tumor microenvironment and improve the CAR-T/NK cells. T/NK cell anti-tumor effect.
  • the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a new cell system, and constructs a CAR-T/NK immunotherapy system according to the method of the present invention, which can only secrete effector molecules inside the tumor , Resist the influence of the tumor microenvironment, even change the tumor microenvironment, improve the therapeutic effect and safety of CAR technology, and reduce toxicity.
  • Figure 1 is a diagram of the mechanism of tumor microenvironment-mediated immune suppression
  • FIG. 2 is a schematic diagram of four generations of CAR-T cell technology in the prior art
  • Figure 3 is the classic pathway and other pathways of NFAT activation
  • Figure 4 shows the apoptosis of target cells mediated by granzyme B through the classic GranB/perforin pathway
  • Figure 5 is a diagram of the carrier and action mode in the cell activation-dependent secretion system of the present invention.
  • Figure 6 is the application of cell activation-dependent secretion system in NK cells
  • Figure 7 shows the expression of peripheral blood T cell CAR and membrane-bound nano-luciferase (MbNanoluc) detected by flow cytometry after lentiviral transduction;
  • FIG. 8 is the application of cell activation-dependent secretion system in T cells
  • Figure 9 shows the application of the cell activation-dependent secretion system in NK cells to secrete mXCL1 chemokine.
  • the reagents used in the present invention are all conventional reagents, and the corresponding experimental operation methods and detection methods are all from routine operation guides, such as "Molecular Cloning Experiment Guide” and “Cell Experiment Guide” edited by Cold Spring Harbor Laboratory wait.
  • NK cell expansion medium (PRIME-XV NK Cell CDM) was purchased from Irvine Scientific manufacturer, item number: 91215;
  • Transduction-promoting reagent (Vectofusin-1) was purchased from Miltenyi Biotec manufacturer, article number: 130-111-163;
  • Biotin-Protein L was purchased from Genscript manufacturer, article number: M00097;
  • Streptavidin-APC (secondary antibody) was purchased from BD Biosciences manufacturer, article number: 554067;
  • Anti-HA tag-AlexaFluor 488 antibody was purchased from Biolegend manufacturer, catalog number: 901509;
  • RPMI1640 complete medium was purchased from Gibco manufacturer, article number: C11875500BT;
  • Granzyme B ELISA kit was purchased from RayBiotech manufacturer, article number: ELH-GZMB;
  • Luciferase reagent was purchased from Promega manufacturer, product number: N1120.
  • the pHIV plasmid was purchased from Addgene, Cat. No.: 21373.
  • the invention constructs a cell activation-dependent secretion system by taking nano luciferase as a model of bioactive molecules.
  • a self-cleaving 2A polypeptide was used to construct a multi-gene co-expression vector regulated by a single promoter.
  • the membrane-bound nanoluciferase (Membrane-bound Nanoluciferase, MbNanoLuc) region connected to the cut 2A polypeptide (P2A) and the CAR region, the MbNanoLuc region is also called the secretory element domain.
  • the CAR of NK cells contains scFv, CD8hinge, NKG2D TM, 2B4 IC and CD3 ⁇ , and the MbNanoLuc region includes a leader sequence (LS), HAtag and NanoLuc, enzyme-cleaved substrate polypeptide (Substrate peptide, SP) and CD8hinge&TM.
  • NK cells were transduced by lentiviral technology to co-express CAR and membrane-bound nanoluciferase (Membrane-bound Nanoluciferase, MbNanoLuc) containing Gran B-specific cleavage substrate polypeptide; After CAR-NK cells are activated through the binding of CAR to the target antigen, Gran B secreted to the extracellular space in the final stage of killing target cells cleaves MbNanoLuc into free nano-luciferase, which can be measured by detecting the activity of free nano-luciferase Whether the surrounding Gran B can effectively cut MbNanoLuc.
  • MbNanoLuc membrane-bound nanoluciferase
  • the enzyme cleavage substrate polypeptide can be AIEFDSGC (IEFD), ASGIEPDSGGSC (IEPD) and VGPDFGR (VGPDF). Meanwhile, MbNanoluc (NS-nanoluc) without specific substrate polypeptide (ie, nonsense polypeptide NS) was used as a control.
  • the cDNA fragment of the CAR cDNA fragment and the secretory element was connected to the expression frame downstream of the pHIV plasmid EF1a promoter by restriction endonuclease digestion and ligase connection or homologous recombination method.
  • NK cell expansion medium PRIME-XV NK Cell CDM
  • ELISA was used to detect the level of granzyme B in the culture supernatant.
  • CAR-NK cells and Raji cells were co-cultured in 250 ⁇ L RPMI1640 complete medium containing 10% FBS at a ratio of 1:1 (CAR-NK92 cells: 5 ⁇ 10 4 cells).
  • the level of granzyme B was detected by ELISA experiment, and the specific operation was carried out according to the instructions of the granzyme B ELISA kit.
  • Nano luciferase activity detection After co-cultivating cells according to the above method, centrifuge, suck out 100 ⁇ L supernatant, and transfer to a white opaque 96-well microplate, add 100 ⁇ L of The luciferase reagent was mixed well, and after waiting for 3 minutes, the luminescence value was measured on the machine.
  • NK92 cells human NK cell lines (NK92 cells) were transduced with a lentiviral vector expressing CD19-CAR and a membrane-bound nanoluciferase (MbNanoluc) containing a granzyme B digestion substrate polypeptide,
  • MbNanoluc membrane-bound nanoluciferase
  • target cells Raji cells
  • CAR-NK92 CAR-NK92 in the control group (NS-nanoluc) without specific substrate polypeptides (nonsense polypeptides).
  • the co-culture of cells did not increase the activity of nano-luciferase in the culture supernatant, suggesting that granzyme B could not cut the nano-luciferase expressed on the CAR-NK cell membrane, while MbNanoluc containing specific substrate polypeptide
  • adding target cells to CAR-NK92 cells for co-culture can significantly increase the activity of nano-luciferase in the culture supernatant.
  • the co-expression vector included a T cell-specific CAR region and a membrane-bound nano-luciferase (Membrane- bound Nanoluciferase, MbNanoLuc) region, the MbNanoLuc region is also known as the secretory element domain.
  • the CAR of T cells contains scFv, CD8hinge, CD8TM, 4-1BB IC, CD3 ⁇ structure, MbNanoLuc region including leader sequence (LS), HAtag and NanoLuc, enzyme cleavage substrate polypeptide (Substrate peptide, SP) and CD8hinge&TM.
  • T cells were transduced by lentivirus technology to co-express CAR and membrane-bound Nanoluciferase (MbNanoLuc) containing Gran B-specific cleavage substrate polypeptide; CAR-T cells interacted with the target antigen through CAR After binding and activation, Gran B secreted to the extracellular space in the final stage of killing target cells cuts MbNanoLuc into free nano-luciferase, and the activity of free nano-luciferase can be detected to determine whether Gran B secreted to the surrounding can effectively cut Cut MbNanoLuc.
  • MbNanoLuc membrane-bound Nanoluciferase
  • the enzyme cleavage substrate polypeptide can be AIEFDSGC (IEFD), ASGIEPDSGGSC (IEPD) and VGPDFGR (VGPDF). Meanwhile, MbNanoluc (NS-nanoluc) without specific substrate polypeptide (ie, nonsense polypeptide NS) was used as a control.
  • CD19-CAR co-expression vector CD19-scFv, CD8hinge, NKG2D TM, 2B4 IC, and CD3 ⁇ were sequentially connected in series to synthesize cDNA fragments expressing the CAR structure, and LS, HA tag, NanoLuc, SP, CD8 hinge&TM were sequentially connected in series to synthesize the cDNA fragments of the secretory element (MbNanoLuc) to express the CAR structure
  • the cDNA fragment of and the cDNA fragment of the secretory element (MbNanoLuc) are connected by P2A; wherein, the cDNA sequence of NanoLuc can be found at https://www.addgene.org/browse/sequence/177060.
  • the CAR cDNA fragment and the cDNA fragment of the secretory element were connected to the expression frame downstream of the pHIV plasmid EF1a promoter by restriction endonuclease digestion and ligase ligation or homologous recombination.
  • ELISA was used to detect the level of granzyme B in the culture supernatant.
  • CAR-T cells (1x105 cells) and Raji cells were co-cultured in 250 ⁇ L RPMI1640 complete medium containing 10% FBS at a ratio of 1:1. After 12 hours of culture, centrifuged, 100 ⁇ L supernatant was collected for ELISA test to detect granzyme B The specific operation was carried out according to the instructions of the granzyme B ELISA kit.
  • Nano luciferase activity detection After co-cultivating cells according to the above method, centrifuge, suck out 100 ⁇ L of supernatant, and transfer to a white opaque 96-well microtiter plate, add 100 ⁇ L of The luciferase reagent was mixed well, and after waiting for 3 minutes, the luminescence value was measured on the machine.
  • Example 1 construct a CAR co-expression vector for cell activation-dependent secretion of mXCL1, select IEFD as the cleavage substrate polypeptide, and connect the mXCL1 sequence region and the transmembrane region as shown in Figure 9A; After successfully constructing the plasmid, package the lentivirus, measure the virus titer and transduce NK92 cells;
  • CD19 CAR-NK92 cell activation-dependent secreted mXCL1 level by ELISA CD19 CAR-NK cells were first cultured in RPMI1640 complete medium for 24 hours, and then CD19 CAR-NK92 cells and Raji cells were mixed at a ratio of 1:1. Ratio (CAR-NK92 cells: 3x10 4 cells) co-cultured in 250 ⁇ L RPMI1640 complete medium containing 10% FBS, after 12 hours of culture, centrifuged, collected 100 ⁇ L supernatant for ELISA test to detect the level of mXCL1, specific operation according to mXCL1 ELISA kit (RayBio Mouse Lymphotactin ELISA Kit) instructions;
  • the system of the present invention can realize the controllable secretion of biologically active molecules dependent on cell activation, and provides a new solution for cell signal transduction research, protein molecular expression research, and tumor immunotherapy after various receptor ligands are bound.

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Abstract

提供了一种细胞激活依赖性分泌系统，该系统包括细胞和载体，该载体导入细胞后表达能够分泌至细胞膜或细胞周边的分泌元件，细胞激活后产生的切割分子切割所述分泌元件。还提供了一种CAR-T/NK细胞激活依赖性免疫治疗系统，CAR-T/NK细胞在肿瘤局部激活后产生的颗粒酶切割分泌元件产生改变肿瘤微环境的效应分子，提高肿瘤免疫治疗效果以及安全性。

Description

一种细胞激活依赖性分泌系统及应用
本申请要求于2021年08月11日提交中国专利局、申请号为“202110917623.5”、发明名称为“一种细胞激活依赖性分泌系统及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及细胞生物学、免疫治疗领域，更具体的本发明是一种细胞激活依赖性分泌系统及其应用。
背景技术
目前，以免疫检查点抑制剂为代表的肿瘤免疫治疗为部分肿瘤患者带来显著生存获益，然而大部分患者对其反应不佳或存在原发性抵抗 [1]。因此，全球正在加快研发各种新的免疫治疗手段。嵌合抗原受体(Chimeric antigen receptor,CAR)免疫细胞，作为一种基因工程化免疫细胞治疗方法，能够精准、快速、高效地杀灭肿瘤，成为一种极具前景的新型肿瘤免疫治疗方法 [2]。目前，经CAR修饰的T细胞(CAR-T)已经在血液恶性肿瘤中获得惊人的疗效，然而，因肿瘤特异性抗原靶点的选择、受限的肿瘤内浸润、肿瘤内免疫抑制性微环境、肿瘤细胞异质性这些因素限制了基于CAR的基因工程化免疫细胞在恶性实体瘤治疗中的临床应用 [3]。
肿瘤微环境(tumor microenvironment,TME)是肿瘤组织内肿瘤细胞、间质细胞、免疫细胞及所分泌的活性介质共同构成的免疫抑制性局部内环境(参见图1)。TME不仅限制了CAR工程化改造免疫细胞(比如CAR-T或CAR-NK细胞)的浸润，而且可以使得已经浸润至肿瘤内的效应免疫细胞不能有效发挥功效。研究显示，TME内肿瘤细胞或间质细胞表达的免疫检查点分子(例如PD-1/PD-L1、NKG2A/HLA-E、TIGIT/CD155)、可溶性免疫抑制性分子(如TGF-β、PGE2)、免疫抑制性代谢产物(如乳酸、ADO)等损害了T细胞或NK细胞的活化及细胞毒性 [4]。因此，人们研究发现：如果CAR修饰的免疫细胞(比如CAR-T或NK细胞，CAR-T/NK)联合静脉给予免疫抑制性分子特异性抗体(如抗-NKG2A抗体)或促进免疫细胞增殖及抗肿瘤活性的细胞因子(比如IL-2、IL-15等)治疗，则可以逆转TME的免疫抑制作用，提高CAR-T/NK细胞的抗肿瘤活性。另一 种方法则是通过基因工程化改造NK细胞，使其直接表达免疫检查点阻断性抗体或免疫激活性细胞因子，以促进CAR-T/NK的持久性及抗肿瘤活性。然而，无论是静脉给药还是NK细胞自身过表达分泌型免疫激活性因子，均可能导致全身系统性毒性，比如免疫检查点抑制剂相关的自身免疫性毒性，IL-15相关的出血事件及胃肠道缺血坏死 [5]。
众所周知，CAR-T(Chimeric antigen receptor T cell，嵌合抗原受体T细胞)疗法，是指通过基因修饰技术，将带有特异性抗原识别结构域及T细胞激活信号的遗传物质转入T细胞，使T细胞直接与肿瘤细胞表面的特异性抗原相结合而被激活，通过释放穿孔素、颗粒酶B等直接杀伤肿瘤细胞，同时还通过释放细胞因子募集人体内源性免疫细胞杀伤肿瘤细胞，从而达到治疗肿瘤的目的，而且还可形成免疫记忆T细胞，从而获得特异性的抗肿瘤长效机制。颗粒酶B(Granzyme B，Gran B)是目前发现在CTLs和NK细胞中诱导靶细胞凋亡活力最强的颗粒酶，在介导靶细胞凋亡中起核心作用。Gran B进入靶细胞后切割含Ile/Val、Glu/Met/Gln、Pro/Xaa和Asp氨基酸序列(P4-P1)的特异性底物，引发靶细胞凋亡(参见图4) [6]。除了进入靶细胞介导靶细胞凋亡外，分泌至CTLs或NK细胞周围的大量GranB同样能有效地剪切含特异性氨基酸序列的分子。目前，CAR-T细胞疗法已经发展出四代(参见图2)。第一代CAR-T结构只含抗原识别信号和CD3ζ信号激活域，体内增殖能力有限；因此，第二代和第三代CAR-T分别加入了一个和两个共刺激分子，以提高T细胞增殖活性、细胞毒性及体内存活时间。第四代CAR-T细胞技术，又称为TRUCK T细胞技术，是通过在CAR-T细胞内转入含有活化T细胞核因子(nuclear factor of the activated T cell，NFAT)，转录相应元件的细胞因子表达载体，促使CAR-T细胞受靶抗原刺激活化后能在肿瘤局部分泌目的细胞因子。在生理或病理条件下，T细胞受体(TCR)与特异性抗原结合后激活CD3胞浆区免疫受体酪氨酸激活模体(ITAM)介导的活化信号，引起胞外Ca 2+内流，激活丝氨酸/苏氨酸钙调磷酸酶(Calcineurin)，从而使NFAT活化并转位至核内，作用于细胞因子启动子上游的NFAT反应元件，诱导多种细胞因子的表达(参见图3) [7-8]；值得注意的是，除了TCR信号外，其他活化性刺激也可导致NFAT的活化(参见图3) [8]。第四代CAR-T技术 正是利用这一原理，将含有NFAT反应元件的细胞因子表达盒导入CAR-T细胞，CAR-T细胞与靶细胞抗原结合并活化后，即可诱导目的细胞因子的表达及分泌(比如IL-12，参见图2) [9]。
TURCK CAR-T技术的目的是通过CAR-T细胞与靶抗原结合并活化后，诱导目的细胞因子表达及分泌，从而尽可能使细胞因子聚集在肿瘤组织内部，减少外周分泌产生的系统性毒性。然而，这种TRUCK T细胞技术存在很大的安全风险 [8]，主要是由于NFAT启动子的“渗漏”、T细胞自身TCR(内源性TCR)触发的NFAT活化及CD3信号非依赖性NFAT激活(参见图6) [9-11]。一项早期临床试验利用诱导型表达IL-12的TRUCK技术工程化肿瘤浸润性淋巴细胞(TILs)过继性治疗黑色素瘤，尽管呈现出明显的治疗性优势，然而产生了大量的毒性包括肝损害、高热及血流动力学紊乱 [12]；患者血清中检测出高水平的IL-12，提示IL-12分泌进入循环系统产生了系统性毒性。此外，CAR表达和细胞因子诱导型表达盒所用的启动子必须为两个不同的启动子，为了避免诱导型表达盒被强组成型CAR启动子转激活，需进行先后两次病毒载体转导来获得CAR及诱导型表达盒分别位于不同基因组位点的TRUCK CAR-T细胞，这种反复的病毒转导不仅增加了CAR-T细胞工艺的复杂性，同时损害了T细胞活性 [8,13]。综上，第四代CAR技术并不能真正意义上实现靶向递送免疫活性分子，无法满足临床上对CAR-T细胞治疗安全性的需求，从而制约了它们在临床中的应用。
因此，理想的技术是使CAR-T/NK细胞能在肿瘤组织局部分泌所需的效应分子，达到改善肿瘤微环境，实现有效的免疫细胞浸润及高效杀灭肿瘤细胞的目的，同时降低或避免活性分子分泌入血介导的全身系统性毒性。
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发明内容
本发明的目的在于提供一种新的细胞系统，其可以用于各种受体配体结合后的细胞信号传导研究、蛋白分子表达研究等各个方面，该系统本发明称之为细胞激活依赖性分泌系统。
为了达到上述目的，本发明采用以下技术方案：
本发明的一个方案中，提供了一种细胞激活依赖性分泌系统，所述系统包括细胞和载体，所述载体导入细胞后表达能够分泌至细胞膜或细胞周边的分泌元件，细胞激活后产生的切割分子切割所述分泌元件。
本发明的一个方案中，提供了一种细胞激活依赖性分泌系统，所述系统包括细胞和载体，所述载体导入细胞后表达能够分泌至细胞膜或细胞周边的分泌元件，细胞激活后产生的切割分子切割所述分泌元件。所述分泌元件与细胞特异性激活受体在同一个载体上表达然后分泌元件分泌至细胞膜或细胞周边
本发明的一个方案中，分泌元件与细胞特异性激活受体在同一个载体上表达，所述载体包括由配体结合域、跨膜结构域、胞内结构域组成的细胞特异性激活受体区，以及分泌元件结构域。
本发明的一个方案中，所述分泌元件与细胞特异性激活受体在同一个载体上由单一启动子驱动共表达，所述载体包括由配体结合域、跨膜结构域、胞内结构域组成的细胞特异性激活受体区，以及分泌元件结构域。
在本发明的一个方案中，所述分泌元件与细胞特异性激活受体在同一个载体上由不同启动子驱动表达，所述载体包括由配体结合域、跨膜结构 域、胞内结构域组成的细胞特异性激活受体区，以及分泌元件结构域。
本发明的一个方案中，分泌元件结构域包括前导序列区、生物活性分子区、切割分子底物区，所述生物活性分子区与切割分子底物区连接；所述生物活性分子区表达的生物活性分子在细胞被激活后发挥生物活性分子作用；所述切割分子底物区表达切割分子底物，在细胞被激活后产生的切割分子切割所述切割分子底物释放与之连接的生物活性分子。
本发明的一个方案中，分泌元件结构域包括前导序列区、生物活性分子区、切割分子底物区及跨膜区，所述生物活性分子区与切割分子底物区连接；所述生物活性分子区表达的生物活性分子在细胞被激活后发挥生物活性分子作用；所述切割分子底物区表达切割分子底物，在细胞被激活后产生的切割分子切割所述切割分子底物释放与之连接的生物活性分子。
本发明的一个方案中，胞内结构域与分泌元件结构域通过可以在单个载体上表达多个多顺反子的元件连接；优选所述元件为表达自剪切多肽的自剪切元件或内部核糖体进入位点(IRES)元件。
本发明的一个方案中，所述细胞特异性激活受体为细胞内源性基因或外源性基因表达产生的受体分子，其与配体结合后激活细胞产生切割分子；优先所述细胞特异性激活受体为嵌合抗原受体(CAR)或T细胞抗原受体(TCR)。
本发明的一个方案中，所述细胞特异性激活受体为嵌合抗原受体CAR包括用于T细胞的CAR，或用于NK细胞的CAR；其中用于NK细胞的CAR含有scFv、CD8hinge、NKG2D TM、2B4 IC、CD3ζ结构；用于T细胞的CAR含有scFv、CD8hinge、CD8 TM、4-1BB IC、CD3ζ结构。
本发明的一个方案中，所述切割分子为酶或具有切割活性的蛋白，优选所述切割分子为剪切酶、更优选所述切割分子为颗粒酶，颗粒酶可以是颗粒酶A、颗粒酶B、颗粒酶H、颗粒酶K、颗粒酶B、颗粒酶M。在一个实施例中剪切酶为caspase，优选caspase-8、caspase-10、caspase-3、caspase-7。
本发明的一个方案中，所述生物活性分子为蛋白质分子、多肽中的一种或多种；所述蛋白质分子包括抗体、抗体活性片段、细胞因子、受体、配体等；
本发明的一个方案中，生物活性分子包括促进被激活的细胞增殖或分化或活性改变的分子、促进靶细胞凋亡或活性改变的分子、对其他细胞有募集作用或能改变局部微环境的分子。
在本发明的一个方案中，生物活性分子是细胞因子/趋化因子，如XCL1、IFNα、IL-12、IL-2、IL-7、IL-15等细胞因子，抗体或抗体片段、多肽片段、小分子抑制剂等。
本发明的一个方案中，所切割分子底物为酶切底物多肽；所述胞内结构域与分泌元件结构域通过表达自剪切多肽的自剪切区域连接；被激活的细胞为免疫细胞，优选T细胞、NK细胞、γδT、NKT、巨噬细胞、树突状细胞或B细胞，酶切底物多肽序列含有IEFD、IEPD、VGPD或IEAD。在一个实施例中，酶切底物多肽序列含有ELQTDG、DEVDR、RIEADS。
在本发明的一个方案中，本发明还提供了一种核酸，所述核酸包括细胞特异性激活受体区和分泌元件结构域，所述核酸插入到表达载体中，进而导入细胞，在细胞中表达所述细胞特异性激活受体和分泌元件，分泌元件被分泌表达至细胞膜或细胞周边，细胞激活后产生的切割分子切割所述分泌元件。
在本发明的一个方案中，本发明还提供了一种表达载体，所述载体导入细胞后表达能够分泌至细胞膜或细胞周边的分泌元件，细胞激活后产生的切割分子切割所述分泌元件；优选所述载体包括细胞特异性激活受体区和分泌元件结构域；更优选所述载体包括嵌合抗原受体区和分泌元件结构域。
在本发明的一个方案中，本发明还提供了一种宿主细胞，所述细胞含有前述表达载体。在一个实施例中，所述宿主细胞为免疫细胞，优选T细胞、NK细胞、γδT细胞、NKT细胞、巨噬细胞、树突状细胞或B细胞。
在本发明的一个方案中，本发明还提供了一种药物组合物，所述药物组合物包括前述任一种细胞激活依赖性分泌系统，核酸、表达载体或宿主细胞，和药学上可接受的载体、稀释剂或赋形剂。
在本发明的一个方案中，本发明还提供了一种细胞激活依赖性分泌系统的应用，所述系统为前述任一所述的系统，所述系统用于制备细胞制剂或细胞药物。所述细胞制剂或细胞药物能与靶细胞上的配体结合而被激活， 释放生物活性分子，所述生物活性分子促进被激活的细胞增殖或分化或活性改变、促进靶细胞凋亡或活性改变、或对其他细胞有募集作用。
在本发明的一个方案中，系统中的细胞特异性激活受体为嵌合抗原受体CAR时，位于系统中的细胞表面表达的嵌合抗原受体CAR与靶细胞表面抗原结合后，系统中的细胞被激活产生切割分子，切割分子切割与嵌合抗原受体CAR共表达的位于细胞膜上或细胞周边中的分泌元件，位于分泌元件中的生物活性分子被释放，释放的生物活性分子进一步促进系统中细胞对于靶细胞的杀伤作用或维持或提高系统中细胞的效应功能及其扩增能力、持续能力。
在本发明的一个方案中，本发明所述系统中细胞是免疫细胞，优选T细胞、NK细胞、γδT细胞、NKT细胞、巨噬细胞、树突状细胞或B细胞。
在本发明的一个方案中，本发明所述的制剂/药物，制剂为液体制剂，药物为注射剂。
在本发明的一个方案中，本发明所述系统、宿主细胞、药物组合物，还可以同其他具有治疗、辅助治疗的药物、制剂、器械、疗法共同使用以增强效果。
在本发明的一个方案中，本发明所述靶细胞为肿瘤细胞，所述肿瘤细胞来自于实体瘤或体液瘤，优选来自实体瘤。所述实体瘤包括头颈部恶性肿瘤、胸部及纵隔肿瘤、消化系统肿瘤、泌尿生殖系统恶性肿瘤、骨与软组织肿瘤、乳腺癌、淋巴瘤、皮肤恶性肿瘤、黑色素瘤、中枢神经系统肿瘤中的一种或多种。
在本发明的一个方案中，本发明所述靶细胞为肿瘤细胞，所述肿瘤为咽癌、喉癌、口腔癌、食管癌、胸腺癌、肺癌、胰腺癌、结肠癌、结直肠癌、胃癌、肝癌、肾癌、膀胱癌、前列腺癌、卵巢癌、宫颈癌、子宫内膜癌、睾丸癌、骨肉瘤、平滑肌肉瘤、乳腺癌、淋巴瘤、皮肤恶性肿瘤、黑色素瘤、脑膜瘤、胶质瘤中的一种或多种。
本发明还提供了一种细胞激活依赖性分泌系统的应用，所述系统为前述任一所述的系统，所述系统可以用于制备试剂盒。
本发明还提供了一种试剂盒，所述试剂盒包括前述的系统、核酸、载体或宿主细胞中的一种或多种。在一个实施例中，所述试剂盒还包括试剂 容器、试剂盒使用说明和/或包装材料等。
本发明的一个方案中，细胞特异性激活受体和分泌元件不是位于同一段核酸或者载体上，他们可以分别位于不同的核酸或者载体上，在使用时，含有所述细胞特异性激活受体区和分泌元件结构域的核酸或载体被分别转导至同一个细胞中并表达至细胞膜或细胞周边区域。位于细胞膜上的细胞特异性激活受体被外源配体激活后，细胞产生切割分子切割分泌元件释放分泌元件中的生物活性分子。
本发明的一个方案中，本发明的一个方案中，细胞特异性激活受体和分泌元件不是位于同一段核酸或者载体上，他们可以分别位于不同的核酸或者载体上，在使用时，含有所述细胞特异性激活受体区和分泌元件结构域的核酸或载体被分别转导至同一个细胞中。细胞特异性激活受体区表达的细胞特异性激活受体定位到细胞膜上。细胞特异性激活受体与外源配体结合激活后，所述受体能够在胞内激活表达分泌元件的载体，分泌元件被分泌至细胞膜或细胞周边，进而，细胞特异性激活受体与外源配体结合激活后产生的切割分子切割分泌元件释放分泌元件中的生物活性分子。
在本发明的一个方案中，生物活性分子具有促进含有细胞特异性激活受体的细胞对于带有配体的靶细胞的杀伤作用，或维持或提高含有细胞特异性激活受体的细胞的效应功能及其扩增能力、持续能力，改善肿瘤微环境、诱导系统性抗肿瘤免疫反应。
在本发明的一个方案中，本发明提供了一种细胞激活依赖系分泌系统，所述系统包括导入细胞后分泌表达至细胞膜或细胞周边的分泌元件，细胞激活后产生的切割分子切割所述分泌元件。所述细胞免疫细胞，优选T细胞、NK细胞、γδT细胞、NKT细胞、巨噬细胞、树突状细胞或B细胞。
在本发明的一个方案中，细胞激活依赖性分泌系统的制备方法包括：
1)利用自剪切多肽构建单一启动子或多启动子调控的多基因表达载体；
2)利用细胞转染技术转导T细胞或NK细胞让其共表达CAR及含有剪切酶底物多肽的膜结合型生物活性分子；
所述自剪切多肽为自剪切2A多肽；所述剪切酶为颗粒酶B；所述剪切酶底物多肽序列含有IEFD，IEPD或VGPD。
在本发明的一个方案中，CAR-T/NK细胞通过CAR与靶抗原结合，在杀伤靶细胞的最后阶段分泌至细胞外的剪切酶切割含有酶切底物多肽的膜结合型生物活性分子，释放生物活性分子。
在本发明的一个方案中，自剪切多肽可以是T2A、P2A、E2A、F2A等在表达后能够自剪切的蛋白肽；
在本发明的一个方案中，所述载体选自下组：质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、DNA、RNA、转座子、或其组合；优选的本发明的载体为慢病毒载体。
与第四代TRUCK技术利用的是T活化初始阶段启动的基因转录活性显著的不同，本发明开发的细胞激活依赖性分泌系统是利用CAR-T/NK细胞激活后发挥靶细胞杀伤效应最后阶段分泌的Gran B的酶切活性(即在效应的终末阶段)，来切割释放生物活性分子，避免了非杀伤性活化信号产生的非目的性分泌，因此，分泌的生物活性分子更能在CAR-T/NK细胞杀伤靶细胞的肿瘤组织内聚集，有利于在肿瘤局部微环境中发挥作用，从而避免了大量非目的性分泌介导的全身系统性毒性。此外，因为NK细胞不存在T细胞受体(TCR)，采用本发明技术方案构建的工程化CAR-NK细胞避免了因内源性TCR被其他抗原(非肿瘤抗原)激活所诱导的非肿瘤组织内分泌，进一步提高安全性。因此，相比于第四代TRUCK CAR-T技术，本发明构建的激活依赖分泌型CAR-T/NK细胞在免疫治疗中更大可能地实现安全地靶向性递送生物活性分子至肿瘤微环境，达到降低分泌性活性分子毒副反应的目的。利用本发明的技术方案可望将CAR-T/NK细胞改造成安全高效的“微型制药工厂”，在肿瘤内产生所需的生物活性分子来克服肿瘤微环境介导的免疫抑制，提高CAR-T/NK细胞抗肿瘤效应。
应理解，在本发明范围内中，本发明的上述各个技术方案以及各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。
与现有技术相比，本发明具有以下有益效果：本发明提供一种新的细胞系统，按照本发明的方法构建CAR-T/NK免疫治疗系统，其能够做到仅在肿瘤内部分泌效应分子，抵抗肿瘤微环境的影响，甚至改变肿瘤微环境，提高CAR技术的治疗效果以及安全性，降低毒性。
附图说明
图1为肿瘤微环境介导免疫抑制的机制图；
图2为现有技术中的四代CAR-T细胞技术示意图；
图3为NFAT活化的经典途径及其他途径；
图4为颗粒酶B通过经典的GranB/穿孔素途径介导的靶细胞凋亡；
图5为本发明细胞激活依赖性分泌系统中的载体与作用模式图；
图6为细胞激活依赖性分泌系统在NK细胞中的应用；
图7为流式检测转导慢病毒转导后外周血T细胞CAR及膜结合型纳米荧光素酶(MbNanoluc)的表达；
图8为细胞激活依赖性分泌系统在T细胞中的应用；
图9为细胞激活依赖性分泌系统在NK细胞中应用于分泌mXCL1趋化因子。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下实施例。除特殊说明外，本发明所使用的试剂均为常规试剂，相应的实验操作方法和检测方法均来自于常规操作指南，例如冷泉港实验室编著的《分子克隆实验指南》、《细胞实验指南》等。
本发明的相关试剂：
NK细胞扩增培养基(PRIME-XV NK Cell CDM)购自Irvine Scientific厂家，货号:91215；
促转导试剂(Vectofusin-1)购自Miltenyi Biotec厂家，货号:130-111-163；
Biotin-Protein L购自Genscript厂家，货号:M00097；
Streptavidin-APC(二抗)购自BD Biosciences厂家，货号:554067；
anti-HA tag-AlexaFluor 488抗体购自Biolegend厂家，货号:901509；
Raji细胞来源CELLCOOK；
RPMI1640完全培养基购自Gibco厂家，货号:C11875500BT；
颗粒酶B ELISA试剂盒购自RayBiotech厂家，货号:ELH-GZMB；
Figure PCTCN2021143120-appb-000002
荧光素酶试剂购自Promega厂家，货号:N1120。
所述pHIV质粒购自Addgene厂家，货号:21373。
实施例1基于NK细胞的细胞激活依赖性分泌系统的构建
本发明以纳米荧光素酶为生物活性分子的模型构建了一种细胞激活依赖性分泌系统。
首先，利用自剪切2A多肽构建单一启动子调控的多基因共表达载体,如图5中的A所示，单一启动子调控的多基因共表达载体包括NK细胞特异性CAR区域，以及通过自剪切2A多肽(P2A)与CAR区域连接的膜结合型纳米荧光素酶(Membrane-bound Nanoluciferase,MbNanoLuc)区域，MbNanoLuc区域也被称为分泌元件结构域。NK细胞的CAR含有scFv、CD8hinge、NKG2D TM、2B4 IC和CD3ζ，MbNanoLuc区域包括前导序列(LS)、HAtag和NanoLuc、酶切底物多肽(Substrate peptide,SP)和CD8hinge&TM。
其次，通过检测游离纳米荧光素酶活性来测定分泌到周围的Gran B是否能有效剪切MbNanoLuc，进而验证系统的可行性。如图5中的B所示，通过慢病毒技术转导NK细胞让其共表达CAR及含有Gran B特异性剪切底物多肽的膜结合型纳米荧光素酶(Membrane-bound Nanoluciferase,MbNanoLuc)；CAR-NK细胞通过CAR与靶抗原结合活化后，在杀伤靶细胞的最后阶段分泌至细胞外的Gran B把MbNanoLuc剪切成游离纳米荧光素酶，可以通过检测游离纳米荧光素酶活性来测定分泌到周围的Gran B是否能有效剪切MbNanoLuc。酶切底物多肽(Substrate peptide,SP)可以是AIEFDSGC(IEFD)，ASGIEPDSGGSC(IEPD)和VGPDFGR(VGPDF)。同时，将不含特异性底物多肽(即无义多肽NS)的MbNanoluc(NS-nanoluc)用作对照。
具体的：
(1)多基因共表达载体CD19-CAR共表达载体的构建。依次串联CD19-scFv、CD8hinge、NKG2D TM、2B4 IC和CD3ζ合成表达CAR结构的cDNA片段，依次串联LS、HA tag、NanoLuc、SP、CD8 hinge&TM合成分泌元件(MbNanoLuc)的cDNA片段，表达CAR结构的cDNA片段与分泌元件(MbNanoLuc)的cDNA片段通过P2A连接；其中，NanoLuc的cDNA序列可参见https://www.addgene.org/browse/sequence/177060。通过限制性内切酶酶切及连接酶连接或同源重组方法将CAR cDNA片段和 分泌元件的cDNA片段连接至pHIV质粒EF1a启动子下游的表达框内。
(2)NK92细胞培养及慢病毒转导。NK92细胞培养于含500IU/ml IL-2的NK细胞扩增培养基(PRIME-XV NK Cell CDM)中，利用离心法(1000g，水平离心1小时)并添加促转导试剂Vectofusin-1(Miltenyi Biotec)进行慢病毒转导(MOI＝10)。
(3)流式细胞检测转导效率。在转导72小时后流式检测转导效率，CAR的表达使用Biotin-Protein L及Streptavidin-APC(二抗)进行染色,利用anti-HA tag-AlexaFluor 488抗体检测分泌元件MbNanoLuc在细胞膜上的表达。
(4)ELISA检测培养上清中颗粒酶B的水平。CAR-NK细胞与Raji细胞按1:1比例(CAR-NK92细胞:5x10 4细胞数)共培养于250μL含10％FBS RPMI1640完全培养基中，培养12小时后，离心，收集100μL上清液进行ELISA实验检测颗粒酶B的水平，具体操作按颗粒酶B ELISA试剂盒说明书进行。
(5)纳米荧光素酶活性检测。按上述方法共培养细胞后，离心，吸出100μL上清液，并转移到白色不透明96孔酶标板中，各孔添加100μL的
Figure PCTCN2021143120-appb-000003
荧光素酶试剂并混匀，等待3分钟后，上机测量发光值。
实验结果：
如图6中的A所示，将表达CD19-CAR和含有颗粒酶B酶切底物多肽的膜结合型纳米荧光素酶(MbNanoluc)的慢病毒载体转导人NK细胞株(NK92细胞)，表达不同MbNanoluc慢病毒载体在NK92细胞中的转导效率为38％-85％之间。
如图6中的B所示，与CAR-T细胞一致，在不含特异性底物多肽(无义多肽)的对照组(NS-nanoluc)中，将靶细胞(Raji细胞)与CAR-NK92细胞共培养没有升高培养上清中纳米荧光素酶的活性，提示颗粒酶B并不能剪切表达在CAR-NK细胞膜上的纳米荧光素酶，而在含表达特异性底物多肽的MbNanoluc的CAR-NK92细胞组，添加靶细胞至CAR-NK92细胞中共培养可以显著增加培养上清中纳米荧光素酶的活性。
如图6中的C所示，通过分析添加靶细胞与未添加靶细胞的CAR-NK92细胞培养上清中纳米荧光素酶活性的比值，显示在CAR-NK 细胞上颗粒酶B同样对于IEFP底物多肽具有显著更高的特异性剪切效率(P<0.001)。
实施例2基于T细胞的细胞激活依赖性分泌系统的构建
首先，构建单一启动子调控的多基因共表达载体，共表达载体包括T细胞特异性CAR区域，以及通过自剪切2A多肽(P2A)与CAR区域连接的膜结合型纳米荧光素酶(Membrane-bound Nanoluciferase,MbNanoLuc)区域，MbNanoLuc区域也被称为分泌元件结构域。T细胞的CAR含有scFv、CD8hinge、CD8TM、4-1BB IC、CD3ζ结构，MbNanoLuc区域包括前导序列(LS)、HAtag和NanoLuc、酶切底物多肽(Substrate peptide,SP)和CD8hinge&TM。
其次，通过检测游离纳米荧光素酶活性来测定分泌到周围的Gran B是否能有效剪切MbNanoLuc，进而验证系统的可行性。通过慢病毒技术转导T细胞让其共表达CAR及含有Gran B特异性剪切底物多肽的膜结合型纳米荧光素酶(Membrane-bound Nanoluciferase,MbNanoLuc)；CAR-T细胞通过CAR与靶抗原结合活化后，在杀伤靶细胞的最后阶段分泌至细胞外的Gran B把MbNanoLuc剪切成游离纳米荧光素酶，可以通过检测游离纳米荧光素酶活性来测定分泌到周围的Gran B是否能有效剪切MbNanoLuc。酶切底物多肽(Substrate peptide,SP)可以是AIEFDSGC(IEFD)，ASGIEPDSGGSC(IEPD)和VGPDFGR(VGPDF)。同时，将不含特异性底物多肽(即无义多肽NS)的MbNanoluc(NS-nanoluc)用作对照。
具体的：
(1)多基因共表达载体CD19-CAR共表达载体的构建。依次串联CD19-scFv、CD8hinge、NKG2D TM、2B4 IC和CD3ζ等合成表达CAR结构的cDNA片段，依次串联LS、HA tag、NanoLuc、SP、CD8 hinge&TM合成分泌元件(MbNanoLuc)的cDNA片段，表达CAR结构的cDNA片段与分泌元件(MbNanoLuc)的cDNA片段通过P2A连接；其中，NanoLuc的cDNA序列可参见https://www.addgene.org/browse/sequence/177060。通过限制性内切酶酶切及连接酶连接或同源重组方法将CAR cDNA片段和分泌元件的cDNA片段连接至pHIV质粒EF1a启动子下游的表达框内。
(2)T细胞培养及慢病毒转导。人T细胞利用人T细胞分选试剂盒(STEMCELL Technologies)从人外周血PBMC中分离出来，分选后的T细胞培养于T细胞扩增培养基(STEMCELL Technologies)，并添加T Cell TransAct(Miltenyi Biotec)进行刺激，细胞被刺激3～4天后利用离心法(1000g，水平离心1小时)并添加促转导试剂Vectofusin-1(Miltenyi Biotec)进行慢病毒转导(MOI＝10)。
(3)流式细胞检测转导效率。在转导72小时后流式检测转导效率，CAR的表达使用Biotin-Protein L及Streptavidin-APC(二抗)进行染色,利用anti-HA tag-AlexaFluor 488抗体检测分泌元件MbNanoLuc在细胞膜上的表达。
(4)ELISA检测培养上清中颗粒酶B的水平。CAR-T细胞(1x105细胞数)与Raji细胞按1:1比例共培养于250μL含10％FBS RPMI1640完全培养基中，培养12小时后，离心，收集100μL上清液进行ELISA实验检测颗粒酶B的水平，具体操作按颗粒酶B ELISA试剂盒说明书进行。
(5)纳米荧光素酶活性检测。按上述方法共培养细胞后，离心，吸出100μL上清液，并转移到白色不透明96孔酶标板中，各孔添加100μL的
Figure PCTCN2021143120-appb-000004
荧光素酶试剂并混匀，等待3分钟后，上机测量发光值。
实验结果：
如图7所示，流式细胞检测显示人T细胞慢病毒转导效率在40-85％。
如图8中的A所示，与未转导慢病毒的T细胞(Untransduced)比较，CAR-T细胞与Raji细胞共培养后显著升高了培养上清中分泌的颗粒酶B(Granzyme B,GzmB)的水平。
如图8中的B所示，通过检测培养上清中纳米荧光素酶的活性，发现在不含特异性底物多肽(无义多肽)的对照组(NS-nanoluc)中，将靶细胞(Raji细胞)与CAR-T细胞共培养没有升高培养上清中纳米荧光素酶的活性，提示颗粒酶B并不能把表达在CAR-T细胞膜上的纳米荧光素酶有效地剪切下来，而在含表达特异性底物多肽的MbNanoluc的CAR-T细胞组，添加靶细胞至CAR-T细胞中共培养可以显著增加培养上清中纳米荧光素酶的活性，其中IEFD组升高的水平最为显著。
如图8中的C所示，通过分析添加靶细胞与未添加靶细胞的CAR-T 细胞培养上清中纳米荧光素酶活性的比值，发现颗粒酶B对于IEFP底物多肽具有更高的剪切效率。
结论：结合实施例1～2的实验结果显示，CAR-NK/T共表达载体在免疫细胞中成功表达，并且T/NK细胞被靶细胞(Raji)上的配体CD19激活后产生的颗粒酶B能够切割膜结合型纳米荧光素酶，释放荧光素酶到细胞培养上清中，如果把纳米荧光素酶换成能改造肿瘤免疫微环境的生物活性分子(比如细胞因子或抗体)，就可突破肿瘤微环境免疫抑制这一瓶颈问题，提高CAR-T/NK细胞实体瘤治疗的效果。
实施例3基于NK细胞的细胞激活依赖性分泌趋化因子系统的构建
构建激活依赖性分泌小鼠XCL1(mXCL1)趋化因子的CAR-NK细胞体系。
(1)首先，根据实施例1所述载体构建方法，构建细胞激活依赖性分泌mXCL1的CAR共表达载体，选择IEFD作为切割底物多肽，连接mXCL1序列区及跨膜区如图9A所示；成功构建质粒后，包装慢病毒，测定病毒滴度后转导NK92细胞；
(2)流式检测NK92细胞慢病毒转导效率：在转导72小时后流式检测转导效率，CAR的表达使用Biotin-Protein L及Streptavidin-APC(二抗)进行染色,利用anti-HA tag-AlexaFluor 488抗体检测mXCL1在细胞膜上的表达；
(3)ELISA法检测CD19 CAR-NK92细胞激活依赖性分泌mXCL1的水平：先将CD19 CAR-NK细胞培养于RPMI1640完全培养基24小时，然后再将CD19 CAR-NK92细胞与Raji细胞按1:1比例(CAR-NK92细胞:3x10 4细胞数)共培养于250μL含10％FBS RPMI1640完全培养基中，培养12小时后，离心，收集100μL上清液进行ELISA实验检测mXCL1的水平，具体操作按mXCL1 ELISA试剂盒(RayBio Mouse Lymphotactin ELISA Kit)说明书进行；
实验结果：如图9B所示，NK92细胞慢病毒转导效率为85.7％。如图9C所示，NK92细胞及CD19 CAR-NK92在没有靶细胞的培养上清中不能检测到mXCL1的分泌，NK92与CD19+Raji细胞共培养后，同样不能检测到mXCL1的分泌，而CD19 CAR-NK92细胞与CD19+Raji细胞 共培养后，可以检测到显著的mXCL1分泌(P<0.001)，提示mXCL1的分泌依赖于CAR与靶细胞上CD19抗原的结合介导的细胞激活，体现了我们建立的细胞激活依赖性分泌技术得到了有效应用。
综上，通过实施例1-3证实了本发明构建的系统、载体、工程化细胞的有效性。本发明的系统能够做到生物活性分子细胞激活依赖性的可控分泌，为各种受体配体结合后的细胞信号传导研究、蛋白分子表达研究，肿瘤免疫治疗提供了全新的解决方案。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (27)

  1. 一种细胞激活依赖性分泌系统，其特征在于，所述系统包括细胞和载体，所述载体导入细胞后表达能够分泌至细胞膜或细胞周边的分泌元件，细胞激活后产生的切割分子切割所述分泌元件。
  2. 根据权利要求1所述的细胞激活依赖性分泌系统，其特征在于，所述分泌元件与细胞特异性激活受体在同一个载体上表达，然后分泌元件分泌至细胞膜或细胞周边；或，利用不同的载体在同一个细胞中分别表达细胞特异性激活受体和分泌元件，然后分泌元件分泌至细胞膜或细胞周边。
  3. 根据权利要求2所述的细胞激活依赖性分泌系统，其特征在于，所述分泌元件与细胞特异性激活受体在同一个载体上表达，所述载体包括由配体结合域、跨膜结构域、胞内结构域组成的细胞特异性激活受体区，以及分泌元件结构域。
  4. 根据权利要求3所述的细胞激活依赖性分泌系统，其特征在于，所述分泌元件结构域包括前导序列区、生物活性分子区和切割分子底物区；所述生物活性分子区与切割分子底物区连接；所述生物活性分子区表达的生物活性分子在细胞被激活后发挥生物活性分子作用；所述切割分子底物区表达切割分子底物，在细胞被激活后产生的切割分子切割所述切割分子底物，释放与之连接的生物活性分子。
  5. 根据权利要求4所述的细胞激活依赖性分泌系统，其特征在于，所述分泌元件结构域还包括跨膜区。
  6. 根据权利要求2～5任一项所述的细胞激活依赖性分泌系统，其特征在于，所述细胞特异性激活受体为细胞内源性基因或外源性基因表达产生的受体分子，其与配体结合后激活细胞产生切割分子。
  7. 根据权利要求6所述的细胞激活依赖性分泌系统，其特征在于，所述细胞特异性激活受体为嵌合抗原受体CAR或T细胞抗原受体TCR。
  8. 根据权利要求7所述的细胞激活依赖性分泌系统，其特征在于，所述嵌合抗原受体CAR包括用于T细胞的CAR或用于NK细胞的CAR；所述用于NK细胞的CAR含有scFv、CD8hinge、NKG2D TM、2B4 IC、CD3ζ结构；所述用于T细胞的CAR含有scFv、CD8hinge、CD8 TM、4-1BB IC、CD3ζ结构。
  9. 根据权利要求6所述的细胞激活依赖性分泌系统，其特征在于，所述切割分子为酶或具有切割活性的蛋白。
  10. 根据权利要求9所述的细胞激活依赖性分泌系统，其特征在于，所述切割分子为颗粒酶。
  11. 根据权利要求4所述的细胞激活依赖性分泌系统，其特征在于，所述生物活性分子为蛋白质、多肽、核酸中的一种或多种；所述蛋白质为抗体、抗体活性片段、细胞因子、受体或配体；所切割分子底物为酶切底物多肽；所述胞内结构域与分泌元件结构域通过在单个载体上表达多个多顺反子的元件连接。
  12. 根据权利要求11所述的细胞激活依赖性分泌系统，其特征在于，所述元件为表达自剪切多肽的自剪切元件或内部核糖体进入位点IRES元件。
  13. 根据权利要求4或11所述的细胞激活依赖性分泌系统，其特征在于，所述生物活性分子包括促进被激活的细胞增殖或分化或活性改变的分子、促进靶细胞凋亡或活性改变的分子、对其他细胞有募集作用或能改变局部微环境的分子。
  14. 根据权利要求13所述的细胞激活依赖性分泌系统，其特征在于，所述被激活的细胞为免疫细胞；所述免疫细胞包括T细胞、NK细胞、γδT细胞、NKT细胞、巨噬细胞、树突状细胞或B细胞。
  15. 根据权利要求13所述的细胞激活依赖性分泌系统，其特征在于，所述靶细胞为肿瘤细胞。
  16. 根据权利要求11所述的细胞激活依赖性分泌系统，其特征在于，所述酶切底物多肽含有IEFD、IEPD、VGPD或IEAD。
  17. 一种核酸，其特征在于，所述核酸包括细胞特异性激活受体区和分泌元件结构域。
  18. 一种载体，其特征在于，所述载体导入细胞后表达能够分泌至细胞膜或细胞周边的分泌元件，细胞激活后产生的切割分子切割所述分泌元件。
  19. 根据权利要求18所述的载体，其特征在于，所述载体包括细胞特异性激活受体区和分泌元件结构域。
  20. 根据权利要求19所述的载体，其特征在于，所述载体包括嵌合抗原受体区和分泌元件结构域。
  21. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求18～20任意一项所述的载体。
  22. 根据权利要求21所述的宿主细胞，其特征在于，所述宿主细胞为免疫细胞；所述免疫细胞为T细胞、NK细胞、γδT细胞、NKT细胞、巨噬细胞、树突状细胞或B细胞。
  23. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1～16任一所述的细胞激活依赖性分泌系统或者权利要求17所述的核酸或者权利要求18～20任意一项所述的载体或权利要求21或22所述的宿主细胞，和药学上可接受的载体、稀释剂或赋形剂。
  24. 权利要求1～16任一所述的细胞激活依赖性分泌系统的应用，其特征在于，所述细胞激活依赖性分泌系统用于制备细胞制剂或细胞药物，或所述细胞激活依赖性分泌系统用于制备试剂盒。
  25. 根据权利要求24所述的应用，其特征在于，所述细胞制剂为液体制剂；所述细胞药物为注射剂。
  26. 权利要求1～16任一所述的细胞激活依赖性分泌系统的制备方法包括：
    1)利用自剪切多肽构建单一启动子或多启动子调控的多基因表达载体；
    2)利用基因转染或病毒转导技术让细胞共表达CAR及含有剪切酶底物多肽的膜结合型生物活性分子；
    所述自剪切多肽为自剪切2A多肽；所述剪切酶为颗粒酶B；所述剪切酶底物多肽序列含有IEFD，IEPD或VGPD。
  27. 根据权利要求26所述的制备方法，其特征在于，所述剪切多肽为T2A、P2A、E2A或F2A。
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