CN114350616A - Immune cell and preparation method and application thereof - Google Patents
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Abstract
The invention discloses an immune cell and a preparation method and application thereof, wherein a promoter is inserted into the upstream of a gene of an alpha E subunit of a CD103 protein in the immune cell, so that the expression of the alpha E subunit is enhanced and stably expressed, and as other subunits of the CD103 protein are highly expressed on the surface of the immune cell and the expression of the alpha E subunit is regulated, the expression of the CD103 is regulated, the expression of the alpha E subunit is enhanced, the expression of the CD103 can be enhanced, and the normal running function of the CD103 can be realized. The presence of the CD103 protein can help the migration of immune cells to epithelial tissues or tumor tissues and enhance the viability of immune cells and the ability to kill tumors. A method for enhancing the expression of endogenous gene is to insert promoters including but not limited to EF1a, CMV and PKG at the upstream of the endogenous gene, so that the expression of the endogenous gene is controlled by the inserted promoters and not controlled by the endogenous promoters, thereby achieving the purpose of enhancing or stabilizing the expression of the endogenous gene.
Description
Technical Field
The invention relates to the technical field of biological cells, in particular to an immune cell for expressing a chimeric antigen receptor and CD103, and a preparation method and application thereof.
Background
The international cell therapy association (interna) in 2012 indicates that biological immune cell therapy has become a fourth means for treating tumors besides surgery, radiotherapy and chemotherapy, and will become a necessary means for treating tumors in the future. The immune cell therapy is to collect peripheral venous blood of a patient, separate peripheral blood mononuclear cells in a GMP laboratory, greatly expand immune effector cells with high-efficiency antitumor activity under the induction of various cytokines, and then return the cells into the body of the patient through intravenous injection, intradermal injection, intervention and the like so as to achieve the purposes of enhancing the immune function of the patient and killing tumor cells.
Chimeric antigen receptor T cells (CAR-T cells) are typically composed of scFv (single-chain variable fragments), transmembrane regions, and intracellular costimulatory signal regions. T cells of a patient are transfected by gene transduction to express a Chimeric Antigen Receptor (CAR). The extracellular scFv of CARs recognizes a specific antigen and then transduces the signal through the intracellular domain, causing activation, proliferation, cytolytic toxicity and secretion of cytokines of T cells, thereby clearing the target cells.
The CAR-T cell treatment technology is more accurate in treatment due to the treatment advantages, more accurate in multi-target, wider in tumor killing range, more durable and more attractive in clinical effect on hematological tumors.
However, CAR-T therapy has some problems, and CAR-T cells have not been as effective in treating solid tumors as hematological tumors, which are important due to inhibition of the tumor microenvironment, problems with homing of T cells to tumor tissue, tumor immune microenvironment, etc.
The CD103 protein expressed on the surface of the T cell is beneficial to the migration and survival of the T cell to epithelial cells and is a marker for the T cell to reside in tissues, but the expression of the CD103 is inhibited due to the existence of an intracellular costimulatory signal region 4-1BB in the CAR-T cell, so that the migration and the standing capacity of the CAR-T cell to tumor tissues are weakened. The study found that the expression of the alpha E subunit of CD103 was inhibited.
According to the existing research data, 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 CAR-T cells containing costimulatory signal region 4-1BB is lower than that of conventional generation CAR (without 4-1BB domain) CD103 protein, so it is suspected that 4-1BB inhibits the expression of CD 103. As shown in fig. 1. Under the stimulation effect of adding Raji cells and TGF-b factors, the expression condition of CD103 protein can be shown, TGF-b can promote CD103 expression, and 4-1BB can antagonize TGF-b signals. As shown in fig. 2.
It is therefore desirable to increase the ability of T cells to migrate to and to lodge in tumor tissue by enhancing the expression of CD 103. CD103 is composed of a plurality of subunits, except for alpha E, the other subunits are highly expressed on the surface of a cell membrane, so that the expression of CD103 can be enhanced by over-expressing the alpha E subunit, but the alpha E subunit protein has a larger molecular structure, and the cell transfer is difficult and low in efficiency by adopting a lentivirus transduction mode or a transposon mode.
Disclosure of Invention
Based on the problems, the invention aims to provide an immune cell which expresses a chimeric antigen receptor and enhances CD103, is beneficial to homing of T cells and survival in a tumor microenvironment, a preparation method and application thereof, and a method for enhancing the expression of endogenous genes of the cell.
The technical scheme of the invention is as follows:
an immune cell, wherein a promoter is added into a gene of an alpha E subunit of a CD103 protein in the immune cell to obtain the alpha E subunit of enhanced CD103, so that the expression of the alpha E subunit is enhanced and stably expressed, and the immune cell expresses the CD103 protein and a chimeric antigen receptor.
In one example, in the immune cell, when the promoter is added, the gene is added at a site upstream of the gene of the α E subunit of the CD103 protein of the immune cell.
In one embodiment, the promoter is one of CMV or a variant thereof, PCK1, and EF1a in an immune cell.
In one embodiment, the promoter is added in a manner of gene editing of any one of criprpr cas9, Talen, ZFN, or transposon in immune cells.
In one embodiment, in an immune cell, the chimeric antigen receptor expressed by the immune cell comprises an antigen binding region, a transmembrane domain, a costimulatory domain, and a stimulatory domain CD3 ζ.
In one embodiment, the chimeric antigen receptor is expressed in an immune cell as a chimeric antigen receptor that targets one or more targets.
In one embodiment, the chimeric antigen receptor is expressed in an immune cell as a first generation CAR cell, a second generation CAR cell, a third generation CAR cell, a fourth generation CAR cell that targets any target.
In one embodiment, the target comprises one or more of CD19, CD20, CD22, claudin18.2, GPC3, GUCY2C, and BCMA in an immune cell.
The immune cells are one or more of peripheral blood T, TIL, NK, NKT and gamma-delta T cells; t cells are preferred.
The invention also relates to a preparation method of the immune cell, which comprises the following steps:
isolation of peripheral blood PBMCs and expansion of immune cells: separating mononuclear cells from peripheral blood, and sorting out immune cells for activation culture; simultaneously adding a slow virus targeting GPC3, transducing the CAR gene to an immune cell genome, and culturing and amplifying;
genetic modification of immune cells: adding a promoter into the upstream of the gene of the alpha E subunit of the immune cell CD103 protein through gene editing to obtain the immune cell stably and highly expressing the CD103 protein;
culturing the modified immune cells: and (3) performing liquid changing treatment on the transformed immune cell culture, culturing the immune cell by using an immune cell complete culture medium, and harvesting the immune cell stably and highly expressing the CD103 protein.
The immune cell provided by the invention can be used for gene expression, and a promoter is added at the upstream of a cell endogenous gene through gene editing, so that the cell endogenous gene is not regulated and controlled by the endogenous promoter, and the stably and highly expressed endogenous gene is obtained.
The invention also provides a preparation method for enhancing the expression of endogenous genes by the alpha E subunit of the CD103 protein, which comprises the following steps:
isolation of peripheral blood PBMCs and expansion of immune cells: separating mononuclear cells from peripheral blood, and sorting out immune cells for activation culture;
genetic modification of immune cells: a promoter is added to the upstream of the gene of the alpha E subunit of the immune cell CD103 protein in a gene editing mode, and the obtained immune cell stably and highly expresses the CD103 protein.
The invention also provides a biological preparation comprising the immune cells and application of the biological preparation in medicaments for preventing and treating tumors and/or cancers. Wherein the biological agent is a pharmaceutically acceptable carrier, diluent or excipient; the tumor is selected from the group consisting of: a hematologic tumor, a solid tumor, or a combination thereof, selected from the group consisting of: 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 group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer or a combination thereof.
Compared with the prior art, the immune cell provided by the invention has the following advantages:
1. the enhancement and stable expression of the CD103 protein can promote immune cells to migrate to tumor tissues and enhance the survival capability and the tumor killing capability of the immune cells;
2. the method for enhancing the CDD103 expression by inserting the promoter has high efficiency, and avoids the defect that the aE subunit has large molecular weight and cannot be stably inserted into a genome for expression by lentivirus, retrovirus, transposon and the like.
3. The requirement of enhancing the expression of endogenous genes with any molecular weight can be met by enhancing the expression of the endogenous genes in a mode of inserting promoters.
Drawings
FIG. 1 shows the expression of CAR-T cells and CD103 protein on the surface of T cells in the prior art;
FIG. 2 is a graph showing the in vitro expression of CD103 protein after stimulation by the addition of target cells to CAR-T cells of the prior art;
FIG. 3 is the structure of the CAR and the structure of CD103 in the examples;
FIG. 4 is a graph of CAR-T cell proliferation expansion in the examples;
FIG. 5 is a histogram of CAR expression in CAR-T cells in the examples;
FIG. 6 is a histogram of CD103 protein expression in CAR-T cells in the examples;
FIG. 7 is a graph of α E expression in CD103 protein in CAR-T cells in the examples;
figure 8 shows CAR-T cells in the examples and different tumor cells at 1: 1. 3: 1. 9: a cell killing rate curve graph after 1-effect target ratio co-culture for 24 h;
figure 9a is a graph of CAR-T cells in the examples with an effective target ratio of 1:1 histogram of IL-2 secretion after 6h of co-culture;
figure 9b is a graph of CAR-T cells in the examples with an effective target ratio of 1:1 after 6h of co-culture, a histogram of INF-gamma secretion;
FIG. 10 is a flow chart of the assay for the expression of E-cadherin by the over-expressed cells Huh7-E-cadherin in the examples;
FIG. 11 is a graph of the effect of mouse tumor size in CAR-T cell animal experiments in the examples;
FIG. 12 is a graph of mouse survival in the CAR-T cell animal assay of the examples;
FIG. 13 is a graph showing T cell infiltration in mouse tumor tissue in animal experiments in examples.
Best mode for carrying out the invention
The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
According to the immune cell provided by the invention, the promoter is added into the alpha E subunit of the CD103 protein of the immune cell, the expression of the alpha E subunit of the CD103 is enhanced, so that the expression of the alpha E subunit is enhanced and stably expressed, and the immune cell expresses the chimeric antigen receptor, as shown in figure 3.
When the immune cell is added with a promoter, the gene locus is the upstream of the gene of the alpha E subunit of the CD103 protein of the immune cell.
In immune cells, the promoter is one of CMV or a variant thereof, PCK1, and EF1 α. The addition mode of the promoter is a mode of editing any one of Crispr cas9, Talen, ZFN or transposon.
The chimeric antigen receptor expressed by the immune cell comprises an antigen binding region, a transmembrane domain, a costimulatory domain, and a stimulatory domain CD3 zeta.
In an immune cell, the chimeric antigen receptor is expressed as a chimeric antigen receptor that targets one or more targets; or the chimeric antigen receptor is expressed as a first generation CAR cell, a second generation CAR cell, a third generation CAR cell, a fourth generation CAR cell targeted to any target.
In the immune cell, the target comprises one or more of CD19, CD20, CD22, Claudin18.2, GPC3, GUCY2C and BCMA.
The immune cells are one or more of peripheral blood T, TIL, NK, NKT and gamma-delta T cells; t cells are preferred.
The invention also relates to a preparation method of the immune cell, which comprises the following steps:
isolation of peripheral blood PBMCs and expansion of immune cells: separating mononuclear cells from peripheral blood, and sorting out immune cells for activation culture; simultaneously adding a slow virus targeting GPC3, transducing the CAR gene to an immune cell genome, and culturing and amplifying;
genetic modification of immune cells: adding a promoter into the upstream of the gene of the alpha E subunit of the CD103 protein of the immune cell through gene editing to obtain the immune cell with the modified alpha E subunit of the CD103 protein;
culturing the modified immune cells: and culturing the modified immune cells by using an immune cell complete culture medium through liquid changing culture, and harvesting the immune cells stably and highly expressing the CD103 protein.
The invention also provides a biological preparation comprising the immune cells and application of the biological preparation in medicaments for preventing and treating tumors and/or cancers. Wherein the biological agent is a pharmaceutically acceptable carrier, diluent or excipient; the tumor is selected from the group consisting of: a hematologic tumor, a solid tumor, or a combination thereof, selected from the group consisting of: 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 group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer or a combination thereof.
The present invention typically describes immune cells of the present invention in detail, taking CAR-T cells as an example. The immune cells of the invention are not limited to the CAR-T cells described above and below, and the immune cells of the invention have the same or similar technical features and benefits as the CAR-T cells described above and below. Specifically, when the immune cell expresses the chimeric antigen receptor CAR, NK cells, NKT cells, TIL, γ - δ T cells are identical to T cells (or T cells can replace NK cells).
In the invention, the chimeric antigen receptors CARs are designed by the following processes:
the first generation of CARs, which had only one intracellular signaling component, CD3 ζ or Fc γ RI molecule, did not achieve good clinical efficacy because it only had one activation domain in the cell, which resulted in only transient T cell proliferation and less cytokine secretion, but did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects;
the second generation CARs introduce a costimulatory molecule such as CD28, 4-1BB, OX40 and ICOS on the basis of the original structure, compared with the first generation CARs, the function is greatly improved, and the persistence of CAR-T cells and the killing capability to tumor cells are further enhanced;
on the basis of the second generation CARs, a plurality of novel immune co-stimulatory molecules such as CD27 and CD134 are connected in series, the third generation CARs and the fourth generation CARs are developed, and double CARs or multiple CARs targeting 2 targets or multiple targets are expressed on the same cell.
The Chimeric Antigen Receptor (CAR) of the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain; wherein the extracellular domain comprises an antigen binding domain; the intracellular domain comprises a costimulatory signaling region and a CD3 zeta chain moiety; a costimulatory signaling region refers to a portion of the intracellular domain that includes a costimulatory molecule; costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens.
The CAR structure provided by the invention is a second generation CAR and consists of a single-chain variable fragment (scFv), a transmembrane domain, a costimulatory domain 4-1BB and a signal domain CD3 zeta; the scFv fragment targets any target, which may be CD19, CD20, CD22, Claudin18.2, GPC3, GUCY 2C.
In the immune cell, the chimeric antigen receptor comprises one or more of a single-chain variable fragment (scFv), a transmembrane domain, a costimulatory domain 4-1BB and/or CD28, a signal domain CD3 zeta and a cytokine gene; and the chimeric antigen receptor is expressed as a chimeric antigen receptor targeting one or more than two targets; for example, the target is an scFv fragment targeting any target; wherein the target comprises one or more of CD19, CD20, CD22, Claudin18.2, GPC3, GUCY2C and BCMA.
The construction of the chimeric antigen receptor CARs plasmid expression frame is realized by adopting a transmission mode, and vectors of the transmission mode comprise DNA, RNA, plasmids, lentiviral vectors, adenovirus, retroviruses, transposons, other gene transfer systems or a combination thereof; preferably the vector is a viral vector. Wherein, the transfer vector is derived from retrovirus such as slow virus in the construction of the expression cassette, and is characterized by long-term and stable integration of target genes into cells; transducible non-proliferating cells, such as hepatocytes; low immunogenicity; the safety is high. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence. The expression vector may be provided to the cell in the form of a viral vector. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. In one embodiment, a lentiviral vector is used.
In the present invention, additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence, another example is elongation growth factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40(SV40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is 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. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.
The immune cells provided by the invention and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the CAR-T cells are present in the formulation at a concentration of 1X 103-1×108Individual cells/ml, more preferably 1X 104-1 ×107Individual cells/ml. In one embodiment, 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; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.
In one embodiment, the immune cells of the invention can undergo robust T cell expansion in vivo and can last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR.
Although the data disclosed herein specifically disclose lentiviral vectors comprising scFv, hinge and transmembrane regions, 4-1BB and CD3 zeta signaling domains, and an inserted promoter for the gene editing transduced CD103 protein, the invention should be construed to include any number of variations to each of the construct components.
Treatable cancers include tumors that are not vascularized or have not substantially vascularized, as well as vascularized tumors. The cancer may comprise a non-solid tumor (such as a hematological tumor, e.g., leukemia and lymphoma) or may comprise a solid tumor. The types of cancer 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, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.
Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid regions. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types 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, ovarian cancer.
The CAR-modified T cell ex vivo procedure of the immune cells of the invention, at least one of the following occurs in vitro prior to administration of the cells into a human: (1) expanding the cell, (2) transducing the CAR structure into the cell, (3) gene editing enhances aE subunit expression and/or (4) cryopreserving the cell. Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from human peripheral blood and genetically modified with vectors expressing the CARs disclosed herein. The CAR-modified cells can be administered to a recipient to provide a therapeutic benefit. And the CAR-modified cell can be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic (syngeneic), or xenogeneic with respect to the recipient.
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. Briefly, a pharmaceutical composition of the invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.
The pharmaceutical composition prepared using the immune cells of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, and by the clinical protocol. When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It can be generally pointed out that: pharmaceutical compositions comprising T cells described herein may be at 1 × 104-1×109Dosage of 1X 10 cells/kg body weight, preferably5-1×107The dose of individual cells/kg body weight was administered. The T cell composition may also be administered multiple times at these doses. Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.
Administration of the biological agents of the invention may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.
In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant treatment modalities, including but not limited to treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or efavirenz therapy for psoriasis patients or other therapy for PML patients. In further embodiments, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an injection of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered pre-or post-surgery. The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Typically, 1X 10 may be administered per treatment or per course of treatment6-1×1010Individual cells/ml are administered to the patient, for example, by intravenous infusion.
The following examples will representatively illustrate immune cells of the present invention in detail, taking CAR-T cells as an example.
Preparation of immune cells
The method comprises the following steps: isolation of peripheral blood PBMC and isolation of T cells
Isolation of mononuclear cells from donor peripheral bloodCells, density gradient centrifugation using ficol, enrichment of T cells with T cell sorting kit (CD3 MicroBeads, human-lysoinvented, 130-097-043), activation of cultured and expanded T cells using magnetic beads coupled with anti-CD3/anti-CD 28; the Medium used was TexMACS GMP Medium (Miltenyi Biotec, 170-2Culturing in a constant temperature incubator.
Step two: cell line culture
This experiment exemplifies the target of GPC3 in solid tumors.
Cell line expressing GPC 3: huh-7 (human hepatoma cells), purchased from ATCC.
Cell line not expressing GPC 3: a549 (human non-small cell lung cancer cells) purchased from ATCC.
Packaging cells: 293T (human embryonic kidney cell line) purchased from ATCC.
Culture in a culture medium: huh-7, A549 and 293T are cultured by using a DMEM medium. All media were supplemented with 10% (v/v) fetal bovine serum.
Step three: CAR structural design and lentiviral packaging
GPC3-CAR structure, i.e. CAR structure targeting GPC3 (glypican 3):
the method constructs a second generation CAR, the core structure of which comprises a secretion signal peptide sequence, scFv of an antibody from anti-GPC3, a CD8/CD28 transmembrane region and 4-1BB which is an intracellular costimulatory signal (the structure is 4-1BB-CD3 zeta).
Cloning GPC3-CAR gene into PHBV lentiviral vector skeleton, placing the PHBV-EF 1 alpha-GPC 3-CAR under a promoter of EF1 alpha (EF-1 alpha) to form PHBVV-EF 1 alpha-GPC 3-CAR, and transferring three plasmids of PHBV-EF 1 alpha-GPC 3-CAR, a lentiviral envelope Plasmid pMD2.G (Addgene, Plasmid #12259) and a lentiviral vector packaging Plasmid psPAX2(Addgene Plasmid #12260) into 293T by using Lipofectamine3000 to prepare a lentiviral complete expression vector; viral supernatants were collected at 48h and 72h, respectively, and concentrated after ultracentrifugation (Merck Millipore); the concentrated virus is ready for infecting T cells.
Step four: CAR-T cell preparation
4.1 Lentiviral infection
After 1 day of activation, isolated and purified primary T cells were infected with lentiviral vectors at MOI (1-10) using step three packaged lentiviruses, and transferred to cell culture flasks at 37 ℃ with 5% CO2Culturing in a constant temperature incubator.
4.2 Gene editing
On day 4 after T cell infection culture, the promoter EF1 α was knocked in by means of criprpr-cas 9 upstream of the α E subunit of CD103 protein in NT (T cell control, no virus infection) cells and/or CAR-T cells, and the process of gene editing included design of grnas, synthesis of templates, and electroporation. Performing electric transfer knockout operation by using PGA, IFN-gamma-gRNA and Cas9 protein, transferring the target gene segment into CAR-T cell by using AAV, transferring the CAR-T cell to a 24-well plate, placing at 37 ℃ and 5% CO2And (5) continuing culturing in the constant-temperature incubator.
4.3 cell culture
After T cell infection, the number of cells is detected by sampling every day on 4 th, 6 th, 8 th, 10 th and 13 th days, the CAR positive rate of the T cells and the expression condition of CD103 protein are detected on 6 th, 10 th and 13 th days, and the culture medium is supplemented every 1-2 days.
After the completion of the T cell infection culture, the CAR-T cells successfully constructed were named GC33-BBz 103CAR-T, and the cell expansion, the expression of CD103, and the expression rate of CAR were compared with each other using T cells (NTs) not infected with lentivirus, i.e., CAR-T cells not subjected to gene editing CD103 protein (GC33-BBz CAR-T) as a control, and the results of the detection are shown in fig. 4 to 7.
The results are shown in fig. 4 and table 1: after 13 days of culture, both CAR-T cells can expand more than 350-fold, and the proliferation rates of GC33-BBz 103CAR-T and GC33-BBz CAR-T are basically consistent.
TABLE 1 cell growth expansion fold number table
As shown in FIG. 5 and Table 2, when the expression of CAR was detected on days 6, 10 and 13 of culture, the CAR positivity of both CAR-T cells was 50-60%, and the expression of CAR from GC33-BBz 103CAR-T was substantially consistent with that of GC33-BBz CAR-T.
TABLE 2 expression efficiency Table of CAR
As shown in fig. 6 and table 3, when CD103 protein expression was detected on days 6, 10, and 13 of culture, GC33-BBz 103CAR-T cells showed an average CD103 protein expression level of 70% or more, which was significantly higher than that of GC33-BBz CAR-T cells with an expression level of 30% or less.
TABLE 3 expression efficiency Table for CD103
It can be seen from the above fig. 5, fig. 6, table 2 and table 3 that the expression of CD103 protein does not affect the expression of CAR in CAR-T cells.
As shown in fig. 7, α 4 and α E share β 7, and α E expression can be increased by overexpression without affecting α 4 and β 7 expression.
(di) CAR-T cell assay detection
1. Cell killing in vitro assay
In vitro killing experiments were performed on three T cells obtained in the four steps. The RTCA DP multifunctional real-time unmarked cell analyzer detects the killing effect of CAR-T cells, A549 cells and Huh-7 cells are respectively subjected to 1:1, 3:1 and 9:1 effect-target ratios, the target cells and the effector cells are incubated for 24h, the killing efficiency is detected and contrasted, and the results are shown in FIG. 8 and Table 4. After the two CAR-T cells are co-cultured with GPC3+ target cells, the in vitro killing efficiency of the two CAR-T cells can reach more than 90%, and the effects are basically consistent.
TABLE 4 table for detecting the killing rate of three T cells
2. Cytokine release assay
And (3) mixing the CAR-T cells obtained in the fourth step with target cells respectively in different effective target ratios, placing the mixture in DEME culture medium, co-culturing for 24h, collecting supernatant, centrifuging, taking the supernatant to detect the release levels of cytokines IL2 and IFN-gamma, and detecting by using an Elisa kit (abbkine, KET6011 and KET6014), wherein the results are shown in FIGS. 9a and 9b, and tables 5 and 6. After co-culture with GPC3+ target cells, figure 9a and table 5 show that GC33-BBz 103CAR-T cells secrete more IL-2, and figure 9b and table 6 show that GC33-BBz 103CAR-T cells secrete relatively lower amounts of IFN- γ.
TABLE 5 IL-2 secretion from NT control cells
| Cell species | NT | GC33-BBz CAR-T | GC33-BBz 103CAR-T |
| Amount of |
3％ | 19.19％ | 24.79％ |
TABLE 6 INF-gamma secretion from INF-gamma control cells
| Cell species | NT | GC33-BBz CAR-T | GC33-BBz 103CAR-T |
| Amount of secretion | 1.459％ | 9.74％ | 7.56％ |
3. Animal testing
3.1 construction of the over-expressed cell line Huh7-E-cadherin
Cloning a basic sequence for expressing cadherin E-cadherin into a lentiviral vector skeleton, designing a plasmid containing the sequence, and transferring the plasmid into 293T by using a three-plasmid packaging system and Lipofectamine3000 to prepare a lentiviral complete expression vector; viral supernatants were collected at 48h and 72h, concentrated by ultracentrifugation (Merck Millipore); the concentrated virus can be used for infecting Huh7, and finally, a Huh7 cell line which expresses E-cadherin and is named as Huh7-E-cadherin is obtained. As shown in FIG. 10, the expression rate of E-cadherin in the Huh7-E-cadherin cell line was 100%.
3.2 mouse assay
Injecting target cells Huh7-E-cadherin subcutaneously into NCG immunodeficient mice for 4-6 weeks, after obvious tumor hard masses appear on the body surfaces of the mice, injecting different CAR-T cells (obtained in step 4) intravenously, and continuously observing the growth condition of the tumor and the survival condition of the mice; the results are shown in FIGS. 11 and 12; the mice were tested for constant follow-up over 40 days and the size of the tumor tissue in the experimental group with GC33-BBz 103CAR-T cells showing more mice could be reduced to 1000mm3The existence of T cells can be detected, which shows that GC33-BBz 103CAR-T cells can better regulate and control E-cadherin-expressing tumor cells and remarkably prolong the growth of miceAnd (7) storing time.
The tumor tissues of the mice are further detected and observed, and the results are shown in fig. 13, the infiltration of GC33-BBz 103CAR-T cells in the tumors is remarkably increased, the morphological change of the T cells in the tumor infiltration is observed under a microscope, the infiltration effect of GC33-BBz 103CAR-T cells is better, and the agglomeration rate of the T cells is obvious.
In the invention, the expression of the alpha E subunit is enhanced by inserting the promoter into the upstream of the alpha E subunit gene of the CD103 protein in immune cells, so that the expression of the alpha E subunit in the CD103 protein is independent of a natural promoter and is not influenced by other signal paths, the chimeric antigen receptor is continuously expressed, and the expression of CD103 is enhanced and stable. The immune cells are easier to migrate to the tumor tissues, are more beneficial to survive in the tumor tissues, can help the immune cells to migrate and survive to the tumor tissues and enhance the tumor killing effect of the immune cells.
It should be understood that the above description is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.
Claims (11)
1. An immune cell, wherein a promoter is inserted into a gene of an α E subunit of a CD103 protein in the immune cell, so that the expression of the α E subunit is enhanced and stably expressed.
2. The immune cell of claim 1, wherein the promoter is added at a gene site upstream of the α E subunit of the CD103 protein of the immune cell.
3. The immune cell of claim 1, wherein the promoter is any one of CMV or a variant thereof, PCK1, and EF1 a.
4. The immune cell of claim 1, wherein the promoter is added by gene editing means of any one of Crispr cas9, Talen, ZFN or transposon.
5. The immune cell of claim 1, wherein the chimeric antigen receptor expressed by the immune cell comprises an antigen binding region, a transmembrane domain, a costimulatory domain, and a stimulatory domain CD3 ζ.
6. The immune cell of claim 5, wherein the chimeric antigen receptor is expressed as a chimeric antigen receptor that targets one or more 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 that targets any target.
7. The immune cell of claim 6, wherein the target comprises one or more of CD19, CD20, CD22, Claudin18.2, GPC3, GUCY2C, and BCMA.
8. A method for producing an immune cell according to any one of claims 1 to 7, comprising the steps of:
isolation of peripheral blood PBMCs and expansion of immune cells: separating mononuclear cells from peripheral blood, and sorting out immune cells for activation culture; simultaneously adding a slow virus targeting GPC3, transducing the CAR gene to an immune cell genome, and culturing and amplifying;
genetic modification of immune cells: adding a promoter into the upstream of the gene of the alpha E subunit of the CD103 protein of the immune cell through gene editing to obtain the immune cell with the modified alpha E subunit of the CD103 protein;
culturing the modified immune cells: and culturing the modified immune cells by using an immune cell complete culture medium through liquid changing culture, and harvesting the immune cells stably and highly expressing the CD103 protein.
9. A method of producing an immune cell according to any one of claims 1 to 7 in which the α E subunit of the CD103 protein enhances the expression of endogenous genes, comprising the steps of:
isolation of peripheral blood PBMCs and expansion of immune cells: separating mononuclear cells from peripheral blood, and sorting out immune cells for activation culture;
genetic modification of immune cells: a promoter is added to the upstream of the gene of the alpha E subunit of the immune cell CD103 protein in a gene editing mode, and the obtained immune cell stably and highly expresses the CD103 protein.
10. A biological agent comprising an immune cell according to any one of claims 1 to 7.
11. Use of the biological agent according to claim 10 for the preparation of a medicament for the prevention and/or treatment of cancer or tumor.
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