WO2023137741A1 - 一种免疫细胞及其制备方法和应用 - Google Patents

一种免疫细胞及其制备方法和应用 Download PDF

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WO2023137741A1
WO2023137741A1 PCT/CN2022/073403 CN2022073403W WO2023137741A1 WO 2023137741 A1 WO2023137741 A1 WO 2023137741A1 CN 2022073403 W CN2022073403 W CN 2022073403W WO 2023137741 A1 WO2023137741 A1 WO 2023137741A1
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cells
immune
cell
immune cells
protein
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French (fr)
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谢海涛
马丽雅
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深圳市先康达生命科学有限公司
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • 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
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention relates to the technical field of biological cells, in particular to an immune cell expressing a chimeric antigen receptor and CD103, a preparation method and application thereof.
  • Immune cell therapy is to collect peripheral venous blood from patients, separate peripheral blood mononuclear cells in a GMP laboratory, and induce a large number of immune effector cells with high anti-tumor activity under the induction of various cytokines, and then reinfuse them into the patient through intravenous, intradermal injection, intervention, etc., to achieve the purpose of enhancing the patient's immune function and killing tumor cells.
  • Commonly used immune cell therapy methods include TCR-T, NK, CAR-T, CIK, DC, TIL, DC+CIK cells, etc.
  • Chimeric antigen receptor T cells are usually scFv (single-chain variable fragment), transmembrane region and intracellular co-stimulatory signal region.
  • the patient's T cells are transfected by gene transduction to express chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the extracellular scFv of CARs can recognize a specific antigen, and then transduce the signal through the intracellular domain, causing T cell activation, proliferation, cytolytic toxicity, and secretion of cytokines, thereby clearing target cells.
  • CAR-T cell therapy technology has attracted more and more attention due to its therapeutic advantages of more accurate treatment, more precise multi-targeting, wider and longer-lasting tumor killing, and it has also achieved remarkable clinical results in hematological tumors.
  • CAR-T therapy there are still some problems in CAR-T therapy.
  • the effect of CAR-T cells in treating solid tumors has not been as good as that of hematological tumors.
  • the important reasons are the suppression of tumor microenvironment, the problem of T cells homing to tumor tissue, and the problem of tumor immune microenvironment.
  • the CD103 protein expressed on the surface of T cells is conducive to the migration and survival of T cells in epithelial cells, and is a marker of tissue-resident T cells.
  • the presence of the intracellular co-stimulatory signal region 4-1BB in CAR-T cells inhibits the expression of CD103, weakening the ability of CAR-T cells to migrate and settle in tumor tissues. The study found that it was due to the inhibition of the expression of the ⁇ E subunit of CD103.
  • the expression level of CD103 protein on the surface of CAR-T cells is lower than that of T cells, and the expression level of CD103 protein in CAR-T cells containing the co-stimulatory signal region 4-1BB is lower than that of the traditional generation of CAR (without 4-1BB domain). Therefore, it is speculated that 4-1BB will inhibit the expression of CD103.
  • Figure 1 Under the stimulation of Raji cells and TGF-b factor, the expression of CD103 protein can be seen that TGF-b can promote the expression of CD103, and 4-1BB can antagonize the TGF-b signal. as shown in picture 2.
  • CD103 is composed of multiple subunits. Except for ⁇ E, the others are highly expressed on the surface of the cell membrane. Therefore, the expression of CD103 can be enhanced by overexpressing the ⁇ E subunit.
  • the protein molecular structure of the ⁇ E subunit is large, and it is difficult and inefficient to transfer into cells by lentiviral transduction or transposon.
  • the problem to be solved by the present invention is to provide an immune cell that expresses chimeric antigen receptor and enhanced CD103, which is beneficial to the homing of T cells and survival in the tumor microenvironment, its preparation method and application, and provides a method for enhancing the expression of endogenous genes in cells.
  • An immune cell adding a promoter to the gene of the ⁇ E subunit of the CD103 protein in the immune cell to obtain an enhanced CD103 ⁇ E subunit, so that the expression of the ⁇ E subunit is enhanced and stably expressed, and the immune cell expresses the CD103 protein and chimeric antigen receptor.
  • the gene site to be added is upstream of the gene of the ⁇ E subunit of the CD103 protein of the immune cells.
  • the promoter in immune cells, is one of CMV or its variants, PCK1 and EF1 ⁇ .
  • the method of adding the promoter is any gene editing method among Crispr cas9, Talen, ZFN or transposon.
  • the chimeric antigen receptor expressed by the immune cells includes an antigen-binding region, a transmembrane domain, a co-stimulatory domain, and a stimulating domain CD3 ⁇ .
  • the chimeric antigen receptor in immune cells, is expressed as a chimeric antigen receptor targeting one or more than two targets.
  • the chimeric antigen receptor in immune cells, is expressed as first-generation CAR cells, second-generation CAR cells, third-generation CAR cells, or fourth-generation CAR cells targeting any target.
  • the target in immune cells, includes one or more of CD19, CD20, CD22, Claudin18.2, GPC3, GUCY2C and BCMA.
  • the above immune cells are one or more of peripheral blood T, TIL, NK, NKT, ⁇ - ⁇ T cells; T cells are preferred.
  • the present invention also relates to a method for preparing the above-mentioned immune cells, comprising the following steps:
  • Isolation of peripheral blood PBMC and expansion of immune cells mononuclear cells were isolated from peripheral blood, and immune cells were sorted out for activation and culture; at the same time, lentiviruses targeting GPC3 were added to transduce the CAR gene into the genome of immune cells, cultured and expanded;
  • a promoter is added to the upstream of the ⁇ E subunit gene of the CD103 protein of the immune cells through gene editing to obtain stable and high-expressing immune cells of the CD103 protein;
  • Transformed immune cell culture For the transformed immune cell culture, change the medium, cultivate immune cells with complete immune cell medium, and harvest immune cells with stable and high expression of CD103 protein.
  • the immune cells provided by the present invention can use the method of gene expression, which adds a promoter upstream of the endogenous gene of the cell through gene editing, so that it is not regulated by the endogenous promoter, and obtains a stable and highly expressed endogenous gene.
  • the present invention also provides a preparation method for enhancing the expression of endogenous genes by the ⁇ E subunit of the above-mentioned CD103 protein, comprising the following steps:
  • Isolation of PBMC from peripheral blood and expansion of immune cells mononuclear cells were isolated from peripheral blood, and immune cells were sorted out for activation and culture;
  • a promoter is added upstream of the ⁇ E subunit gene of immune cell CD103 protein by gene editing, and the obtained immune cells stably and highly express CD103 protein.
  • the present invention also provides a biological preparation comprising the above-mentioned immune cells, and the application of the biological preparation in medicines for preventing and treating tumors and/or cancers.
  • the biological preparation is a pharmaceutically acceptable carrier, diluent or excipient;
  • the tumor is selected from the following group: hematological tumor, solid tumor, or a combination thereof, and the hematological tumor is selected from the following group: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL) or a combination thereof;
  • the solid tumor is selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia Blood disease, kidney cancer, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, lymphoma cancer,
  • the immune cells provided by the present invention have the following advantages:
  • CD103 protein can promote the migration of immune cells to tumor tissues and enhance their viability and ability to kill tumors;
  • the method of enhancing the expression of CDD103 by inserting the promoter is highly efficient, and avoids the disadvantages of stably inserting expression into the genome through lentivirus, retrovirus, transposon, etc. due to the large molecular weight of the aE subunit.
  • Enhancing the expression of endogenous genes by inserting promoters can meet the needs of enhancing the expression of endogenous genes with any large molecular weight.
  • Figure 1 shows the expression of CD103 protein on the surface of CAR-T cells and T cells in the prior art
  • Fig. 2 is the in vitro expression of CD103 protein after being stimulated by adding target cells to CAR-T cells in the prior art
  • Fig. 3 is the structure of CAR and the structure of CD103 in the embodiment
  • Fig. 4 is the CAR-T cell proliferation expansion curve diagram in the embodiment
  • Fig. 5 is the histogram of the expression of CAR in the CAR-T cell in the embodiment.
  • Figure 6 is a histogram showing the expression of CD103 protein in CAR-T cells in the embodiment.
  • Figure 7 is a graph showing the expression of ⁇ E in CD103 protein in CAR-T cells in the embodiment.
  • Figure 8 is a graph of the cell killing rate after co-culture of CAR-T cells and different tumor cells in the embodiment at 1:1, 3:1, and 9:1 effect-to-target ratios for 24 hours;
  • Figure 9a is a histogram of IL-2 secretion after co-culture of CAR-T cells and different tumor cells at an effect-to-target ratio of 1:1 in the embodiment;
  • Figure 9b is a histogram of INF- ⁇ secretion after co-culture of CAR-T cells and different tumor cells at an effect-to-target ratio of 1:1 in the embodiment;
  • Fig. 10 is the detection flow diagram of the overexpression cell Huh7-E-cadherin expressing E-cadherin in the embodiment
  • Figure 11 is a diagram showing the effect of mouse tumor size in the CAR-T cell animal test in the embodiment.
  • Figure 12 is a mouse survival curve in the CAR-T cell animal test in the embodiment.
  • Fig. 13 is a picture of T cell infiltration in mouse tumor tissue in the animal experiments in the examples.
  • a promoter is added to the ⁇ E subunit of the CD103 protein of the immune cells to enhance the expression of the ⁇ E subunit of CD103, so that the expression of the ⁇ E subunit is enhanced and stably expressed, and the immune cells express chimeric antigen receptors, as shown in FIG. 3 .
  • the added gene site is upstream of the gene of the ⁇ E subunit of the immune cell CD103 protein.
  • the promoter is one of CMV or its variants, PCK1 and EF1 ⁇ .
  • the method of adding the promoter Any gene editing method among Crispr cas9, Talen, ZFN or transposon.
  • the chimeric antigen receptor expressed by the immune cells includes an antigen binding region, a transmembrane domain, a co-stimulatory domain and a stimulating domain CD3 ⁇ .
  • the chimeric antigen receptor is expressed as a chimeric antigen receptor targeting one or more than two targets; or the chimeric antigen receptor is expressed as a first-generation CAR cell, a second-generation CAR cell, a third-generation CAR cell, or a fourth-generation CAR cell targeting any target.
  • the targets include one or more of CD19, CD20, CD22, Claudin18.2, GPC3, GUCY2C and BCMA.
  • the above immune cells are one or more of peripheral blood T, TIL, NK, NKT, ⁇ - ⁇ T cells; T cells are preferred.
  • the present invention also relates to a method for preparing the above-mentioned immune cells, comprising the following steps:
  • Isolation of peripheral blood PBMC and expansion of immune cells mononuclear cells were isolated from peripheral blood, and immune cells were sorted out for activation and culture; at the same time, lentiviruses targeting GPC3 were added to transduce the CAR gene into the genome of immune cells, cultured and expanded;
  • a promoter is added to the upstream of the gene of the ⁇ E subunit of the CD103 protein of the immune cell through gene editing to obtain immune cells with the modified ⁇ E subunit of the CD103 protein;
  • Transformed immune cell culture through medium exchange culture, the transformed immune cells are cultivated with the complete medium of immune cells, and the immune cells with stable and high expression of CD103 protein are harvested.
  • the present invention also provides a biological preparation comprising the above-mentioned immune cells, and the application of the biological preparation in medicines for preventing and treating tumors and/or cancers.
  • the biological preparation is a pharmaceutically acceptable carrier, diluent or excipient;
  • the tumor is selected from the following group: hematological tumor, solid tumor, or a combination thereof, and the hematological tumor is selected from the following group: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL) or a combination thereof;
  • the solid tumor is selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia Blood disease, kidney cancer, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, lymphoma cancer,
  • the present invention takes CAR-T cells as an example to representatively describe the immune cells of the present invention in detail.
  • the immune cells of the present invention are not limited to the CAR-T cells described above, and the immune cells of the present invention have the same or similar technical features and beneficial effects as the CAR-T cells described above.
  • NK cells, NKT cells, TIL, ⁇ - ⁇ T cells are equivalent to T cells (or T cells can replace NK cells) when immune cells express chimeric antigen receptor CAR.
  • the first-generation CAR has only one intracellular signaling component CD3 ⁇ or Fc ⁇ RI molecule. Since there is only one activation domain in the cell, it can only cause transient T cell proliferation and less cytokine secretion, but cannot provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical efficacy;
  • the second-generation CARs introduce a co-stimulatory molecule based on the original structure, such as CD28, 4-1BB, OX40, and ICOS. Compared with the first-generation CARs, the function is greatly improved, and the persistence of CAR-T cells and the ability to kill tumor cells are further enhanced;
  • CD27 and CD134 are connected in series to develop into the third-generation and fourth-generation CARs, and there are also double CARs or multi-CARs that target two or more targets on the same cell.
  • the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain; wherein, the extracellular domain includes an antigen-binding domain; the intracellular domain includes a co-stimulatory signal transduction region and a CD3 ⁇ chain part; the co-stimulatory signal transduction region refers to a part of the intracellular domain including co-stimulatory molecules;
  • the CAR structure provided by the present invention is a second-generation CAR, which consists of a single-chain variable fragment (scFv), a transmembrane domain, a co-stimulatory domain 4-1BB and a signaling domain CD3 ⁇ ; the scFv fragment targets any target, and the target can be CD19, CD20, CD22, Claudin18.2, GPC3, GUCY2C.
  • scFv single-chain variable fragment
  • the target can be CD19, CD20, CD22, Claudin18.2, GPC3, GUCY2C.
  • the chimeric antigen receptor includes single-chain variable fragment (scFv), transmembrane domain, co-stimulatory domain 4-1BB and/or CD28, signaling domain CD3 ⁇ and one or more of cytokine genes; and the chimeric antigen receptor is expressed as a chimeric antigen receptor targeting one or more targets; for example, the target is scFv fragment targeting any target; wherein the target includes CD19, CD20, CD22, Claudin18.2, GPC3, GUCY One or more of 2C and BCMA.
  • scFv single-chain variable fragment
  • the target includes CD19, CD20, CD22, Claudin18.2, GPC3, GUCY One or more of 2C and BCMA.
  • the construction of the chimeric antigen receptor CARs plasmid expression cassette is achieved by means of transfer, and the carrier of the transfer method includes DNA, RNA, plasmid, lentiviral vector, adenovirus, retrovirus, transposon, other gene transfer systems, or a combination thereof; the preferred carrier is a viral vector.
  • the delivery vector in the construction of the expression cassette is derived from a retrovirus such as a lentivirus vector, which is characterized by long-term and stable integration of the target gene into cells; it can transduce non-proliferating cells, such as liver cells; low immunogenicity; and high safety.
  • a typical cloning vector contains transcriptional and translational terminators, an initial sequence and a promoter useful for regulating the expression of the desired nucleic acid sequence.
  • Expression vectors can be provided to cells as viral vectors.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • suitable vectors will contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites and one or more selectable markers.
  • retroviruses provide a convenient platform for gene delivery systems. The gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo.
  • lentiviral vectors are used.
  • promoter elements can regulate the frequency of transcription initiation.
  • these are located in the 30-110 bp region upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site.
  • the spacing between promoter elements is often flexible in order to preserve promoter function when elements are inverted or moved relative to one another.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence, another example is elongation growth factor-1 alpha (EF-1 alpha).
  • CMV immediate early cytomegalovirus
  • EF-1 alpha elongation growth factor-1 alpha
  • constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Ruth's sarcoma virus promoter, and human gene promoters such as but not limited to actin promoter, myosin promoter promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention.
  • an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired.
  • inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.
  • the formulation is a liquid formulation.
  • the preparation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione;
  • buffers such as neutral buffered saline, sulfate buffered saline, and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids such as glycine
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • immune cells of the invention can undergo robust in vivo T cell expansion for extended amounts of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific for the antigen-binding domain in the CAR.
  • Treatable cancers include tumors that are not or substantially not vascularized, as well as vascularized tumors.
  • Cancer may include non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) or may include solid tumors.
  • Cancer types treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignancies, such as sarcomas, carcinomas, and melanomas. Also includes adult tumors/cancers and childhood tumors/cancers.
  • Hematological cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroid leukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodge King's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. The different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer and ovarian cancer.
  • CAR-modified T cells of the immune cells of the present invention at least one of the following occurs in vitro before the cells are administered into the human body: (1) expanding the cells, (2) transducing the CAR structure into the cells, (3) gene editing to enhance the expression of the aE subunit and/or (4) cryopreserving the cells.
  • Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells were isolated from human peripheral blood and genetically modified with vectors expressing the CARs disclosed herein. CAR-modified cells can be administered to a recipient to provide a therapeutic benefit. and CAR-modified cells can be autologous to the recipient. Alternatively, the cells may be allogeneic, syngeneic or xenogeneic with respect to the recipient.
  • the CAR-modified T cells of the immune cells of the invention can be administered alone or in combination with other drugs, pharmaceutical compositions, diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione;
  • buffers such as neutral buffered saline, sulfate buffered saline, and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids such as glycine
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • the pharmaceutical composition made using the immune cells of the present invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by such factors as the patient's condition, and the type and severity of the patient's disease, and by the clinical protocol.
  • the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject).
  • a pharmaceutical composition comprising T cells as described herein may be administered at a dose of 1 x 10 4 -1 x 10 9 cells/kg body weight, preferably 1 x 10 5 -1 x 10 7 cells/kg body weight. T cell compositions can also be administered multiple times at these doses.
  • the optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical art by monitoring the patient for signs of disease and adjusting treatment accordingly.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally.
  • the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by i.v. injection.
  • Compositions of T cells can be injected directly into tumors, lymph nodes or sites of infection.
  • cells activated and expanded using the methods described herein, or other methods known in the art to expand T cells to therapeutic levels are administered to the patient in conjunction with (e.g., before, simultaneously with, or after) any number of relevant treatment modalities including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab for MS patients or erfa for psoriasis patients Zizumab therapy or other treatments for patients with PML.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab for MS patients or erfa for psoriasis patients Zizumab therapy or other treatments for patients with PML.
  • T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents.
  • the cell composition of the invention is administered to a patient in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation, use of a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide.
  • a subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery. Dosages administered to a patient for the above treatments will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be implemented according to practice accepted in the art. Usually, 1 ⁇ 10 6 -1 ⁇ 10 10 cells/ml can be administered to the patient for each treatment or each course of treatment, for example, through intravenous infusion.
  • Step 1 Isolation of PBMC from peripheral blood and isolation of T cells
  • Mononuclear cells were isolated from donor peripheral blood, density gradient centrifugation was performed using ficol, and T cells were enriched with a T cell sorting kit (CD3 MicroBeads, human-lyophilized, 130-097-043), and T cells were activated and expanded using magnetic beads coupled with anti-CD3/anti-CD28; the culture medium used TexMACS GMP Medium (Miltenyi Biotec, 170-076-30 9), also containing 10% FBS, 2mM L-glutamine and 100IU/ml rhIL2, etc., and the cells were cultured in a constant temperature incubator at 37°C and 5% CO 2 .
  • a T cell sorting kit CD3 MicroBeads, human-lyophilized, 130-097-043
  • T cells were activated and expanded using magnetic beads coupled with anti-CD3/anti-CD28
  • the culture medium used TexMACS GMP Medium (Miltenyi Biotec, 170-076-30 9), also containing 10% FBS, 2mM L-glutamine and
  • This experiment takes the GPC3 target in solid tumors as an example.
  • Cell line expressing GPC3 Huh-7 (human liver cancer cell), purchased from ATCC.
  • Cell line not expressing GPC3 A549 (human non-small cell lung cancer cells), purchased from ATCC.
  • 293T human embryonic kidney cell line
  • ATCC human embryonic kidney cell line
  • Culture medium Huh-7, A549, and 293T were cultured in DMEM medium. All media were supplemented with 10% (v/v) fetal bovine serum.
  • Step 3 CAR structure design and lentiviral packaging
  • GPC3-CAR structure that is, the CAR structure targeting GPC3 (glypican 3):
  • the method of the present invention constructs a second-generation CAR.
  • the core structure of the CAR includes a secretory signal peptide sequence, scFv from an anti-GPC3 antibody, a CD8/CD28 transmembrane region, and 4-1BB as an intracellular co-stimulatory signal (the structure is 4-1BB-CD3 ⁇ ).
  • the GPC3-CAR gene was cloned into the backbone of the PHBLV lentiviral vector and placed under the promoter of EF1 ⁇ (EF-1 ⁇ ) to form PHBLV-EF1 ⁇ -GPC3-CAR, and the vector packaging plasmid psPAX2 (Addgene Plasmid#12260) three plasmids were transferred into 293T using Lipofectamine3000 to prepare a complete lentiviral expression vector; the virus supernatant was collected at 48h and 72h respectively, and concentrated after ultracentrifugation (Merck Millipore); the concentrated virus can be used to infect T cells.
  • EF1 ⁇ EF-1 ⁇
  • psPAX2 Additional Plasmid#12260
  • the lentivirus packaged in Step 3 was used to infect with the lentivirus vector according to the MOI (1-10), and then transferred to a cell culture flask and cultured in a constant temperature incubator at 37°C and 5% CO 2 .
  • the promoter EF1 ⁇ was knocked in upstream of the ⁇ E subunit of CD103 protein in NT (T cell control, not infected with virus) cells and/or CAR-T cells by means of crispr-cas9.
  • the gene editing process included gRNA design, template synthesis and electroporation.
  • IFN- ⁇ -gRNA and Cas9 protein to perform electroporation knockout operation, using AAV to transfer the target gene fragment into CAR-T cells, and then transfer the CAR-T cells to 24-well plates, and place them in a 37°C, 5% CO2 constant temperature incubator to continue culturing.
  • T cell infection After T cell infection, samples were taken every day to detect the number of cells on the 4th, 6th, 8th, 10th, and 13th day, respectively. On the 6th, 10th, and 13th day, the CAR positive rate of T cells and the expression of CD103 protein were detected, and the culture medium was supplemented every 1-2 days.
  • CAR-T cells After T cell infection and culture, successfully constructed CAR-T cells, named GC33-BBz 103CAR-T, T cells (NT) not infected with lentivirus, that is, CAR-T cells without gene editing of CD103 protein (GC33-BBz CAR-T) were used as the control, and the cell expansion, CD103 expression and CAR expression rate were compared respectively.
  • the detection results are shown in Figures 4 to 7.
  • the expression of CAR was detected on the 6th, 10th, and 13th day of culture respectively.
  • the CAR-positive rates of the two CAR-T cells were both 50%-60%, and GC33-BBz 103CAR-T and GC33-BBz CAR-T have basically the same expression of CAR.
  • CD103 protein As shown in Figure 6 and Table 3, the expression of CD103 protein was detected on the 6th, 10th, and 13th day of culture, and the expression level of CD103 protein in GC33-BBz 103CAR-T cells reached more than 70%, which was significantly higher than that of GC33-BBz CAR-T cells with an expression level of less than 30%.
  • ⁇ 4 and ⁇ E share ⁇ 7, and overexpression can increase the expression of ⁇ E without affecting the expression of ⁇ 4 and ⁇ 7.
  • In vitro killing experiments were performed on the three kinds of T cells obtained in step four.
  • the RTCA DP multifunctional real-time label-free cell analyzer was used to detect the killing effect of CAR-T cells.
  • A549 and Huh-7 cells were used for the effect-to-target ratio of 1:1, 3:1, and 9:1, respectively, and the target cells and effector cells were co-incubated for 24 hours to detect and compare the killing efficiency.
  • the results are shown in Figure 8 and Table 4. After co-culturing with GPC3+ target cells, the in vitro killing efficiency of the two CAR-T cells can reach more than 90%, and the effect is basically the same.
  • the CAR-T cells and target cells obtained in Step 4 were mixed with different effect-to-target ratios, placed in DEME medium, co-cultured for 24 hours, and the supernatant was collected. After centrifugation, the supernatant was taken to detect the release levels of cytokines IL2 and IFN- ⁇ . Elisa kits (abbkine, KET6011, KET6014) were used for detection. The results are shown in Figures 9a, 9b, Table 5 and Table 6.
  • Figure 9a and Table 5 show that GC33-BBz 103CAR-T cells secrete more IL-2
  • Figure 9b and Table 6 show that GC33-BBz 103CAR-T cells secrete a relatively low amount of IFN- ⁇ .
  • the nucleotide sequence expressing cadherin E-cadherin was cloned into the backbone of the lentiviral vector, a plasmid containing the sequence was designed, and the plasmid was transferred into 293T using a three-plasmid packaging system and Lipofectamine3000 to prepare a complete lentiviral expression vector; the viral supernatant was collected at 48h and 72h, and supercentrifuged for concentration (Merck Millipore); the concentrated virus can be used to infect Huh7, and finally obtain the Huh7 cell line overexpressing E-cadherin, which is named as Huh7-E-cadherin. As shown in Figure 10, the expression rate of E-cadherin in the Huh7-E-cadherin cell line was 100%.
  • mice After subcutaneously injecting the target cell Huh7-E-cadherin into NCG immune-deficient mice at 4-6 weeks, after the appearance of obvious tumor lumps on the surface of the mice, different CAR-T cells (obtained in step 4) were injected intravenously, and the tumor growth and survival of the mice were continuously observed; the results are shown in Figure 11 and Figure 12; the mice were continuously tracked and detected within 40 days, and the experimental group using GC33-BBz 103 CAR-T cells showed more tumors.
  • the tumor tissue size of the mice can be reduced to 1000 mm 3
  • the following and the presence of T cells can be detected, indicating that GC33-BBz 103CAR-T cells can better regulate tumor cells expressing E-cadherin and significantly prolong the survival time of mice.
  • the promoter is inserted upstream of the ⁇ E subunit gene of the CD103 protein in the immune cells to enhance the expression of the ⁇ E subunit, so that the expression of the ⁇ E subunit in the CD103 protein does not depend on the natural promoter and is not affected by other signaling pathways, the chimeric antigen receptor is continuously expressed, and the expression of CD103 is enhanced and stable.
  • the immune cells are more likely to migrate to tumor tissue, and are more conducive to survival in tumor tissue, and can help immune cells migrate and survive to tumor tissue, and enhance their tumor-killing effect.

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Abstract

提供了一种免疫细胞及其制备方法和应用，该免疫细胞中CD103蛋白的αE亚基的基因上游插入启动子，使得αE亚基的表达增强且稳定表达。

Description

一种免疫细胞及其制备方法和应用 技术领域
本发明涉及生物细胞技术领域，尤其涉及一种表达嵌合抗原受体和CD103的免疫细胞及其制备方法和应用。
 背景技术
2012年国际细胞治疗协会年会中指出生物免疫细胞治疗已经成为手术、放疗、化疗外的第四种治疗肿瘤的手段，并将成为未来肿瘤治疗必选手段。免疫细胞治疗是采集患者外周静脉血，在GMP实验室内分离外周血单个核细胞，在多种细胞因子诱导下，大量扩增出具有高效抗瘤活性的免疫效应细胞，再通过静脉、皮内注射、介入等回输到患者体内，达到增强患者免疫功能和杀伤肿瘤细胞的目的，常用的免疫细胞治疗手段包括TCR-T,NK, CAR-T,CIK、DC、TIL、DC+CIK 细胞等。
嵌合抗原受体T细胞(CAR-T细胞)通常是scFv(single-chain variable fragment)，跨膜区以及胞内共刺激信号区域组成。通过基因转导的方法转染患者的T细胞，使其表达嵌合抗原受体(CAR)。CARs的胞外段scFv可识别一个特异的抗原，随后通过胞内结构域转导该信号，引起T细胞的活化、增殖、细胞溶解毒性和分泌细胞因子，进而清除靶细胞。
CAR-T细胞治疗技术由于其治疗优势治疗更精准、多靶向更精准，杀瘤范围更广、更持久越来越受到人们的重视，其在血液瘤上也取得了令人瞩目的临床效果。
然而CAR-T治疗还存在一些问题，CAR-T细胞治疗实体瘤的效果一直都不如血液瘤，其重要的原因是肿瘤微环境的抑制，T细胞向肿瘤组织归巢的问题，肿瘤免疫微环境问题等。
T细胞表面表达的CD103蛋白有利于T细胞向上皮细胞中迁移和生存，是组织驻留T细胞的标志，但是由于CAR-T细胞中的胞内共刺激信号区域4-1BB的存在会抑制CD103的表达，减弱了CAR-T细胞向肿瘤组织的迁移和驻足能力。研究发现是由于CD103的αE亚基表达受到抑制所致。
根据已有研究数据表明，CAR-T细胞表面CD103蛋白表达水平相较于T细胞表达更低，且含有共刺激信号区域4-1BB的CAR-T细胞相较于传统的一代CAR（无4-1BB结构域）CD103蛋白的表达水平更低，因此猜测是4-1BB会抑制CD103的表达。如图1所示。在加入Raji细胞和TGF-b因子的刺激作用下，CD103蛋白的表达情况可以看出，TGF-b可以促进CD103表达，4-1BB可以拮抗TGF-b信号。如图2所示。
因此希望通过增强CD103的表达，从而增加T细胞向肿瘤组织迁移和驻足的能力。CD103由多个亚基组成，除了 αE，其他都是高表达在细胞膜表面，因此可以通过过表达 αE亚基的方式来增强CD103表达，但是 αE亚基蛋白分子结构较大，采用慢病毒转导方式或者转座子的方式转入细胞难度较大、效率低。
 技术问题
基于上述问题，本发明所要解决的问题在于提供一种表达嵌合抗原受体和增强型CD103，利于T细胞的归巢及在肿瘤微环境中存活的免疫细胞及其制备方法和应用，并且提供了一种增强细胞内源基因表达的方法。
 技术解决方案
本发明的技术方案如下：
一种免疫细胞，在所述免疫细胞中CD103蛋白的αE亚基的基因中加入启动子，获得增强CD103的αE亚基，使得αE亚基的表达增强且稳定表达，且该免疫细胞表达CD103蛋白和嵌合抗原受体。
一实施例，免疫细胞中，在加入启动子时，加入基因位点为免疫细胞CD103蛋白的αE亚基的基因上游。
一实施例，免疫细胞中，所述启动子为CMV或其变体、PCK1及EF1α中的一种。
一实施例，免疫细胞中，所述启动子的加入方式 Crispr cas9、Talen、ZFN或者转座子中任一种基因编辑的方式。
一实施例，免疫细胞中，所述免疫细胞所表达的嵌合抗原受体包括抗原结合区域、跨膜结构域、共刺激域及刺激域CD3ζ。
一实施例，免疫细胞中，所述嵌合抗原受体表达为靶向一个或两个以上靶点的嵌合抗原受体。
一实施例，免疫细胞中，所述嵌合抗原受体表达为靶向任何靶点的第一代CAR细胞、第二代CAR细胞、第三代CAR细胞、第四代CAR细胞。
一实施例，免疫细胞中，所述靶点包括CD19、CD20、CD22、Claudin18.2、GPC3、GUCY2C及BCMA中的一种或多种。
上述免疫细胞为外周血T、TIL、NK、NKT、γ-δT细胞中的一种或几种；优选T细胞。
本发明还涉及上述免疫细胞的制备方法，包括步骤如下：
外周血PBMC的分离和免疫细胞的扩增：从外周血分离出单个核细胞，并分选出免疫细胞进行激活培养；同时加入靶向GPC3的慢病毒，将CAR基因转导至免疫细胞基因组，培养和扩增；
免疫细胞的基因改造：在免疫细胞CD103蛋白的αE亚基的基因上游通过基因编辑加入启动子，得到稳定高表达CD103蛋白的免疫细胞；
改造后免疫细胞培养：对改造后的免疫细胞培养换液处理，用免疫细胞完全培养基培养免疫细胞，收获稳定高表达CD103蛋白的免疫细胞。
本发明提供的免疫细胞可以通过基因表达的方法，其通过基因编辑在细胞内源基因的上游加入启动子，使其不受内源启动子的调控，得到稳定高表达的内源基因。
本发明还提供上述CD103蛋白的αE亚基增强内源基因表达的制备方法，包括步骤如下：
外周血PBMC的分离和免疫细胞的扩增：从外周血分离出单个核细胞，并分选出免疫细胞进行激活培养；
免疫细胞的基因改造：通过基因编辑方式在免疫细胞CD103蛋白的αE亚基的基因上游加入启动子，得到的免疫细胞稳定高表达CD103蛋白。
本发明还提供一种包括上述免疫细胞的生物制剂，以及该生物制剂在预防、治疗肿瘤和/或癌症药物中的应用。其中，生物制剂为药学上可接受的载体、稀释剂或赋形剂；所述肿瘤选自下组：血液肿瘤、实体肿瘤、或其组合，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)或其组合；所述实体肿瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌或其组合。
 有益效果
与现有技术相比，本发明提供的免疫细胞具有如下优点：
1、CD103蛋白的增强稳定表达可以促进免疫细胞向肿瘤组织迁移并增强其生存能力及杀伤肿瘤能力；
2、通过插入启动子的方式增强CDD103表达的方式效率高，避免 由于aE亚基分子量大，没办法通过慢病毒、逆转录病毒、转座子等方式稳定插入基因组中表达的弊端。
3、通过插入启动子的方式增强内源基因的表达可以满足任意分子量大的内源基因的增强表达的需求。
 附图说明
图1为现有技术中CAR-T细胞和T细胞表面的CD103蛋白的表达情况；
图2为现有技术中的CAR-T细胞中加入靶细胞刺激后CD103蛋白的体外表达情况；
图3为实施例中的CAR的结构和CD103的结构；
图4为实施例中 的CAR-T细胞增殖扩增曲线图；
图5为实施例中的CAR-T细胞中CAR的表达柱状图；
图6为实施例中的CAR-T细胞中CD103蛋白的表达柱状图；
图7为实施例中的CAR-T细胞中CD103蛋白中αE的表达情况图；
图8为实施例中的CAR-T细胞与不同肿瘤细胞分别以1：1、3：1、9：1效靶比共培养24h后的细胞杀伤率曲线图；
图9a为实施例中的CAR-T细胞与不同肿瘤细胞以效靶比1：1共培养6h后IL-2的分泌量柱状图；
图9b为实施例中的CAR-T细胞与不同肿瘤细胞以效靶比1：1共培养6h后INF-γ的分泌量柱状图；
图10为实施例中的过表达细胞Huh7- E-cadherin表达E-cadherin的检测流式图；
图11为实施例中的CAR-T细胞动物试验中小鼠肿瘤大小效果图；
图12为实施例中的CAR-T细胞动物试验中小鼠生存曲线图；
图13为实施例中的动物试验中小鼠肿瘤组织中T细胞浸润图。
 本发明的最佳实施方式
下面结合附图，对本发明的较佳实施例作进一步详细说明。
本发明提供的免疫细胞，在该免疫细胞的CD103蛋白的αE亚基中加入启动子，增强CD103的αE亚基表达，使得αE亚基的表达增强且稳定表达，且该免疫细胞表达嵌合抗原受体，如图3所示。
该免疫细胞在加入启动子时，加入基因位点为免疫细胞CD103蛋白的αE亚基的基因上游。
在免疫细胞中，所述启动子为CMV或其变体、PCK1及EF1α中的一种。所述启动子的加入方式 Crispr cas9、Talen、ZFN或者转座子中任一种基因编辑的方式。
所述免疫细胞所表达的嵌合抗原受体包括抗原结合区域、跨膜结构域、共刺激域及刺激域CD3ζ。
免疫细胞中，所述嵌合抗原受体表达为靶向一个或两个以上靶点的嵌合抗原受体；或者所述嵌合抗原受体表达为靶向任何靶点的第一代CAR细胞、第二代CAR细胞、第三代CAR细胞、第四代CAR细胞。
免疫细胞中，所述靶点包括CD19、CD20、CD22、Claudin18.2、GPC3、GUCY2C及BCMA中的一种或多种。
上述免疫细胞为外周血T、TIL、NK、NKT、γ-δT细胞中的一种或几种；优选T细胞。
本发明还涉及上述免疫细胞的制备方法，包括步骤如下：
外周血PBMC的分离和免疫细胞的扩增：从外周血分离出单个核细胞，并分选出免疫细胞进行激活培养；同时加入靶向GPC3的慢病毒，将CAR基因转导至免疫细胞基因组，培养和扩增；
免疫细胞的基因改造：在免疫细胞CD103蛋白的αE亚基的基因上游通过基因编辑加入启动子，得到CD103蛋白的αE亚基被改造的免疫细胞；
改造后免疫细胞培养：通过换液培养，用免疫细胞完全培养基培养改造后的免疫细胞，收获稳定高表达CD103蛋白的免疫细胞。
本发明还提供一种包括上述免疫细胞的生物制剂，以及该生物制剂在预防、治疗肿瘤和/或癌症药物中的应用。其中，生物制剂为药学上可接受的载体、稀释剂或赋形剂；所述肿瘤选自下组：血液肿瘤、实体肿瘤、或其组合，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)或其组合；所述实体肿瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌或其组合。
本发明以CAR-T细胞为例，代表性地对本发明的免疫细胞进行详细说明。本发明的免疫细胞不限于上下文所述的CAR-T细胞，本发明的免疫细胞具有与上下文所述的CAR-T细胞相同或类似的技术特征和有益效果。具体地，当免疫细胞表达嵌合抗原受体CAR时，NK细胞、NKT细胞、TIL、γ-δT细胞等同于T细胞(或T细胞可替换NK细胞)。
本发明中，嵌合抗原受体CARs的设计经历了以下过程：
第一代CAR只有一个胞内信号组份CD3ζ或者FcγRI分子，由于胞内只有一个活化结构域，因此它只能引起短暂的T细胞增殖和较少的细胞因子分泌，而并不能提供长时间的T细胞增殖信号和持续的体内抗肿瘤效应，所以并没有取得很好地临床疗效；
第二代CARs在原有结构基础上引入一个共刺激分子，如CD28、4-1BB、OX40、ICOS，与一代CARs相比功能有很大提高，进一步加强CAR-T细胞的持续性和对肿瘤细胞的杀伤能力；
在二代CARs基础上串联一些新的免疫共刺激分子如CD27、CD134，发展成为三代和四代CARs，并且还有在同一个细胞上表达靶向2个靶点或者多个靶点的的双CAR或者多CAR等。
本发明的嵌合抗原受体(CAR)包括细胞外结构域、跨膜结构域及细胞内结构域；其中，胞外结构域包括抗原结合结构域；细胞内结构域包括共刺激信号传导区和CD3ζ链部分；共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分；共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子。
本发明提供的CAR结构为第二代CAR，由单链可变片段（scFv）、跨膜结构域、共刺激域4-1BB和信号结构域CD3ζ组成；scFv片段靶向任何靶点，靶点可以是CD19，CD20，CD22，Claudin18.2，GPC3，GUCY2C。
该免疫细胞中，嵌合抗原受体的包括单链可变片段（scFv）、跨膜结构域、共刺激域4-1BB和/或CD28、信号结构域CD3ζ及细胞因子基因中一种或多种；且嵌合抗原受体表达为靶向一个或两个以上靶点的嵌合抗原受体；如，靶点为scFv片段靶向任何靶点；其中，靶点包括CD19、CD20、CD22、Claudin18.2、GPC3、GUCY2C及BCMA中的一种或多种。
嵌合抗原受体CARs质粒表达框的构建是采用传递方式实现的，传递方式的载体包括DNA、RNA、质粒、慢病毒载体、腺病毒、逆转录病、转座子、其他基因转移系统、或其组合；优选载体为病毒载体。其中，表达框的构建中传递载体源于逆转录病毒诸如慢病毒的载体，其特点是长期、稳定的整合目的基因至细胞中；可转导非增殖的细胞，诸如肝细胞；低免疫原性；安全性高。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。表达载体可以以病毒载体形式提供给细胞。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。在一个实施方案中，使用慢病毒载体。
本发明中，额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个被倒置或移动时，保持启动子功能。合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列，另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够当这样的表达是期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
本发明提供的免疫细胞以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方案中，所述制剂为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述CAR-T细胞的浓度为1×10 3 -1×10 8 个细胞/ml，更优地1×10 4-1×10 7 个细胞/ml。在一个实施方案中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的制剂优选配制用于静脉内施用。
在一个实施方案中，本发明的免疫细胞可经历稳固的体内T细胞扩展并可持续延长的时间量。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-修饰T细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。
尽管本文公开的数据具体公开了包括scFv、铰链和跨膜区、4-1BB和CD3ζ信号传导结构域的慢病毒载体，及基因编辑转导的CD103蛋白的插入启动子，但本发明应被解释为包括对构建体组成部分中的每一个的任何数量的变化。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌卵巢癌。
本发明免疫细胞的CAR-修饰T细胞的离体程序，以下中的至少一项在将细胞施用进入人体前在体外发生：（1）扩增细胞，（2）将CAR结构转导入细胞，（3）基因编辑增强aE亚基表达和/或（4）冷冻保存细胞。离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞人外周血中分离并用表达本文公开的CAR的载体进行基因修饰。CAR-修饰的细胞可被施用给接受者，以提供治疗益处。和CAR-修饰的细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
本发明免疫细胞的CAR-修饰的T细胞可被单独施用或与其他药物、药物组合物、稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的组合物优选配制用于静脉内施用。
采用本发明免疫细胞制成的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度，并由临床方案确定。当指出“免疫学上有效量”、 “抗肿瘤有效量”、 “肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可以以1×10 4-1×10 9 个细胞/kg体重的剂量，优选1×10 5 -1×10 7 个细胞/kg体重的剂量施用。T细胞组合物也可以以这些剂量多次施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
本发明的生物制剂的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方案中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方案中，本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方案中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方案中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方案中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方案中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方案中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方案中，扩展的细胞在外科手术前或外科手术后施用。施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 6 -1×10 10 个细胞/ml，通过例如静脉回输的方式，施用于患者。
本发明的实施方式
下述实施例以CAR-T细胞为例，代表性地对本发明的免疫细胞进行详细说明。
（一）免疫细胞的制备
步骤一：外周血PBMC的分离和T细胞的分离
从供体外周血中分离单核细胞，使用ficol进行密度梯度离心，并用T细胞分选试剂盒富集T细胞(CD3 MicroBeads, human - lyophilized，130-097-043)，使用偶联anti-CD3/anti-CD28的磁珠激活培养和扩增T细胞；培养基使用TexMACS GMP Medium(Miltenyi Biotec，170-076-309），还含10％FBS、2mM L-glutamine及100IU/ml rhIL2等，细胞培养均置于37℃，5％CO 2恒温培养箱中培养。
步骤二：细胞系培养
本实验以实体瘤中的GPC3靶点为例。
表达GPC3的细胞系：Huh-7(人肝癌细胞)，购自ATCC。
不表达GPC3的细胞系：A549(人非小细胞肺癌细胞)，购自ATCC。
包装用细胞：293T(人胚肾细胞系)，购自ATCC。
培养基培养：Huh-7、A549、293T使用DMEM培养基培养。所有培养基均添加10％(v/v)胎牛血清。
步骤三：CAR结构设计与慢病毒包装
GPC3-CAR结构，即靶向GPC3（磷脂酰肌醇蛋白聚糖3）的CAR结构：
本发明方法构建了第二代CAR，CAR的核心结构包括分泌信号肽序列、来自anti-GPC3的抗体的scFv、CD8/CD28跨膜区及4-1BB为胞内段共刺激信号（结构为4-1BB-CD3ζ）。
将GPC3-CAR基因克隆至PHBLV慢病毒载体骨架中，置于EF1α(EF-1α)的启动子下，形成PHBLV-EF1α-GPC3-CAR，将PHBLV-EF1α-GPC3-CAR、慢病毒包膜质粒pMD2.G (Addgene，Plasmid#12259)和慢病毒等载体包装质粒psPAX2(Addgene Plasmid#12260)三个质粒，使用Lipofectamine3000转入293T中制备慢病毒完整表达载体；分别在48h和72h收集病毒上清，超速离心后进行浓缩(Merck Millipore)；浓缩后的病毒即可用于感染T细胞。
步骤四：CAR-T细胞制备
4.1慢病毒感染
分离纯化的原代T细胞在激活1天后，利用步骤三包装的慢病毒，按MOI(1-10)进行慢病毒载体感染，然后转移至细胞培养瓶，置于37℃，5％CO 2恒温培养箱中培养。
4.2基因编辑
T细胞感染培养后第4天，通过crispr-cas9的方式在NT（T细胞对照样，不感染病毒）细胞和/或者CAR-T细胞中的CD103蛋白的αE亚基上游敲入启动子EF1α，基因编辑的过程包括gRNA的设计、template的合成及电转。利用PGA、IFN-γ-gRNA和Cas9蛋白进行电转敲除操作，利用AAV将目的基因片段转入CAR-T细胞内，之后将CAR-T细胞转移至24孔板，置于37℃，5％CO 2恒温培养箱中继续培养。
4.3细胞培养
T细胞感染后，分别在第4、6、8、10、13天，每天取样检测细胞数量，第6、10、13天分别检测T细胞的CAR阳性率及CD103蛋白的表达情况，每隔1-2天传代补加培养基。
T细胞感染培养结束后，成功构建了的CAR-T细胞，命名为GC33-BBz 103CAR-T，以不感染慢病毒的T细胞（NT），即未进行基因编辑CD103蛋白的CAR-T细胞（GC33-BBz CAR-T）为对照，分别对比细胞扩增情况、CD103的表达情况和CAR的表达率，检测结果如图4至7所示。
结果如图4和表1所示：培养13天后，两种CAR-T细胞均可扩增350倍以上，可得GC33-BBz 103CAR-T与GC33-BBz CAR-T的增殖速率基本一致。
表1 细胞生长扩增倍数表
如图5及表2所示，分别在培养第6、10、13天检测CAR的表达情况，两种CAR-T细胞的CAR阳性率均在50%~60%，可得GC33-BBz 103CAR-T与GC33-BBz CAR-T的CAR的表达基本一致。
表2 CAR的表达效率表
如图6和表3所示，分别在培养第6、10、13天检测CD103蛋白的表达情况，GC33-BBz 103CAR-T细胞CD103蛋白的表达水平均达到70%以上，显著高于 表达水平30%以下的GC33-BBz CAR-T细胞。
表3 CD103的表达效率表
结合上述图5、图6、表2、表3可知，CD103蛋白的表达不影响CAR-T细胞中CAR的表达情况。
如图7所示，α4和αE共用β7，通过过表达可以增加αE表达但不影响α4和β7的表达。
（二）CAR-T细胞试验检测
1、细胞体外杀伤试验
对步骤四种获得的三种T细胞进行体外杀伤实验。RTCA DP多功能实时无标记细胞分析仪检测CAR-T细胞的杀伤效应，A549、Huh-7细胞分别做1:1、3:1、9:1效靶比，且靶细胞与效应细胞共孵育24h，检测对比杀伤效率，结果如图8和表4所示。与GPC3+靶细胞共培养后，两种CAR-T细胞的体外杀伤效率均可达90%以上，效果基本一致。
表4 三种T细胞杀伤率检测表
2、细胞因子释放检测
将步骤四获得的CAR-T细胞与靶细胞分别以不同效靶比混合，置于DEME培养基中，共培养24h，收集上清，离心后取上清检测细胞因子IL2与IFN-γ的释放水平，采用Elisa试剂盒(abbkine，KET6011、KET6014)进行检测，结果如图9a、9b、表5和表6所示。与GPC3+靶细胞共培养后，图9a和表5显示，GC33-BBz 103CAR-T细胞分泌更多的IL-2，图9b和表6显示，GC33-BBz 103CAR-T细胞分泌IFN-γ的量相对较低。
表5 NT对照样细胞IL-2分泌量
表6 INF-γ对照样细胞INF-γ分泌量
3、动物试验
3.1过表达细胞系Huh7-E-cadherin的构建
将表达钙粘蛋白E-cadherin的碱基序列克隆至慢病毒载体骨架中，设计含有该序列的质粒，将该质粒利用三质粒包装系统和使用Lipofectamine3000转入293T中制备慢病毒完整表达载体；在48h和72h收集病毒上清，超离进行浓缩(Merck Millipore)；浓缩后的病毒即可用于感染Huh7，最终得到过表达E-cadherin的Huh7细胞系，命名为Huh7-E-cadherin。如图10所示，Huh7-E-cadherin细胞系中E-cadherin的表达率为100%。
3.2小鼠试验
将靶细胞Huh7-E-cadherin对4-6周NCG免疫缺陷小鼠进行皮下注射后，待小鼠体表出现明显肿瘤硬块后，静脉注射不同的CAR-T细胞(步骤4获得)，并持续观察肿瘤生长情况及小鼠生存情况；结果如图11和图12所示；在40天内对小鼠进行持续跟踪检测，使用GC33-BBz 103CAR-T细胞的实验组出现更多小鼠的肿瘤组织大小可以减小到1000mm 3以下且能检测到T细胞的存在，说明GC33-BBz 103CAR-T细胞可以更好的调控表达E-cadherin的肿瘤细胞，并显著延长小鼠生存时间。
对小鼠肿瘤组织进行进一步检测和观察，结果如图13所示，GC33-BBz 103CAR-T细胞在肿瘤内的浸润显著增加，且镜下观察到肿瘤浸润中T细胞的形态学变化，GC33-BBz 103CAR-T细胞浸润效果更好，T细胞结团率明显。
 工业实用性
本发明中，免疫细胞中CD103蛋白的αE亚基基因上游插入启动子来增强αE亚基的表达的方式，使CD103蛋白中αE亚基的表达不依赖于天然启动子不受其他信号通路的影响，持续表达嵌合抗原受体，且表达CD103增强并稳定。此免疫细胞更容易向肿瘤组织迁移，且更利于在肿瘤组织中存活，更能帮助免疫细胞向肿瘤组织迁移和生存，并在增强其杀伤肿瘤作用。

Claims (11)

  1. [根据细则26改正 23.02.2022]
    一种免疫细胞，其特征在于，在该免疫细胞中CD103蛋白的aE亚基的基因中插入启动子，使得aE亚基的表达增强且稳定表达。
  2. [根据细则26改正 23.02.2022]
    根据权利要求1所述的免疫细胞，其特征在于，在加入启动子时,加入的基因位点为免疫细胞CD103蛋白的aE亚基的基因上游。
  3. [根据细则26改正 23.02.2022]
    根据权利要求1所述的免疫细胞，其特征在于，所述启动子为CMV或其变体、PCK1及EF1a中的任一种。
  4. [根据细则26改正 23.02.2022]
    根据权利要求1所述的免疫细胞，其特征在于，所述启动子的加入方式为Crisprcas9、Talen、ZFN或者转座子中的任一种基因编辑方式。
  5. [根据细则26改正 23.02.2022]
    根据权利要求1所述的免疫细胞，其特征在于，该免疫细胞所表达的嵌合抗原受体包括抗原结合区域、跨膜结构域、共刺激域及刺激域CD3ζ。
  6. [根据细则26改正 23.02.2022]
    根据权利要求5所述的免疫细胞，其特征在于，所述嵌合抗原受体表达为靶向一个或两个以上靶点的嵌合抗原受体；或者所述嵌合抗原受体表达为靶向任何靶点的第一代CAR细胞、第二代CAR细胞、第三代CAR细胞或第四代CAR细胞。
  7. [根据细则26改正 23.02.2022]
    根据权利要求6所述的免疫细胞，其特征在于，所述靶点包括CD19、CD20、CD22、Claudin18.2、GPC3、GUCY2C及BCMA中的一种或多种。
  8. [根据细则26改正 23.02.2022]
    一种如权利要求1至7任一所述免疫细胞的制备方法，其特征在于，步骤如下:外周血PBMC的分离和免疫细胞的扩增：从外周血分离出单个核细胞,并分选出免疫细胞进行激活培养；同时加入靶向GPC3的慢病毒，将CAR基因转导至免疫细胞基因组，培养和扩增;免疫细胞的基因改造：在免疫细胞CD103蛋白的aE亚基的基因上游通过基因编辑加入启动子，得到CD103蛋白的aE亚基被改造的免疫细胞;改造后免疫细胞培养：通过换液培养，用免疫细胞完全培养基培养改造后的免疫细胞，收获稳定高表达CD103蛋白的免疫细胞。
  9. [根据细则26改正 23.02.2022]
    一种如权利要求1至7任一所述免疫细胞中CD103蛋白的aE亚基增强内源基因表达的制备方法，其特征在于，步骤如下:外周血PBMC的分离和免疫细胞的扩增：从外周血分离出单个核细胞,并分选出免疫细胞进行激活培养;免疫细胞的基因改造：通过基因编辑方式在免疫细胞CD103蛋白的aE亚基的基因上游加入启动子，得到的免疫细胞稳定高表达CD103蛋白。
  10. [根据细则26改正 23.02.2022]
    一种包括如权利要求1至7任一所述免疫细胞的生物制剂。
  11. [根据细则26改正 23.02.2022]
    如权利要求10所述生物制剂在制备预防和/或治疗癌症或肿瘤的药物中应用。
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