CN114854692B - CAR-macrophage and method of making same - Google Patents

CAR-macrophage and method of making same Download PDF

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CN114854692B
CN114854692B CN202210343508.6A CN202210343508A CN114854692B CN 114854692 B CN114854692 B CN 114854692B CN 202210343508 A CN202210343508 A CN 202210343508A CN 114854692 B CN114854692 B CN 114854692B
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CN114854692A (en
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刘韬
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Beijing Mercer Biotechnology Co ltd
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Beijing Mercer Biotechnology Co ltd
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Abstract

The application relates to the technical field of cellular immunity, in particular to a preparation method of CAR-macrophages, which comprises the following steps: constructing a lentivirus containing a nucleotide sequence shown in SEQ ID No. 1; transfection of lentiviruses into CD34 + Obtaining seed cells capable of expressing chimeric antigen receptor with amino acid sequence shown as SEQ ID No.2 in hematopoietic stem cells or induced pluripotent stem cells; and (3) carrying out induced differentiation treatment on the seed cells capable of expressing the chimeric antigen receptor in a macrophage differentiation medium to obtain the CAR-macrophages. The preparation method not only solves the defect that macrophages cannot proliferate, but also can greatly enhance the targeting property of the CAR-macrophages and the phagocytic capacity of tumor cells, so that the CAR-macrophages can be used for treating tumors and have good application prospects in clinic.

Description

CAR-macrophage and method of making same
Technical Field
The application belongs to the technical field of cellular immunity, and particularly relates to a CAR-macrophage and a preparation method thereof.
Background
In recent years, immune cell therapy technology has become one of the most promising research directions following traditional surgical therapies, chemotherapy and radiotherapy. The immune cell therapy is to collect autoimmune cells of human body, culture and amplify and reform the autoimmune cells in vitro to enhance targeting and killing functions, and then to return the autoimmune cells to the body of a patient to achieve therapeutic effects. Currently, immune cell therapies mainly include natural killer cells (NK), chimeric antigen receptor-modified NK cells (CAR-NK), T cell receptor-modified T cells (TCR-T), chimeric antigen receptor-modified T cells (CAR-T), and the like. CAR-T cell therapy has great advantages in cancer treatment, particularly in hematological tumor treatment, however its therapeutic effect on solid tumors is very limited. Unlike hematological tumors, solid tumors are capable of constructing a tumor microenvironment (tumor microenvironment, TME) with immunosuppressive properties, thereby affecting the efficacy of CAR-T therapy. The immunosuppressive changes of TME prevent immune cell trafficking to the focus and infiltration of tumor by T lymphocytes, and a few T cell immune responses that can infiltrate solid tumors may still be limited by immunosuppressive cells or inhibitors in the microenvironment. Based on the advantages of CAR-T cell therapy in clinical applications, researchers have tried and successfully engineered other engineered CAR immune cells in an attempt to overcome the limitations of CAR-T therapy and provide new directions for solid tumor treatment. Among them, CAR-modified macrophages (CAR-M) are considered as a promising cell type.
Macrophages (Macrophages, abbreviation) Belongs to phagocytes, belongs to a monocyte system, is an immune cell widely distributed in whole blood and tissues, participates in the innate immunity and cellular immunity of organisms, and is a key effector cell of an innate immune response. Macrophages are capable of phagocytizing and killing intracellular parasites, bacteria, tumor cells, and cells that are self-senescent and abnormal, playing an important role in the immune defenses, immune homeostasis and immune surveillance of the body; in addition, macrophages can present antigens after phagocytic debris and pathogens to activate lymphocytes or other immune cells to respond to the pathogen. Tumor-associated macrophages (TAM) have both tumor killing and tumor promoting effects. M1 type macrophages are activated by TLR or Th1 cytokines, have high antigen presenting capacity, and also secrete Reactive Oxygen Species (ROS) and pro-inflammatory cytokines, which are correlated with a good prognosis for cancer. In contrast, M2-like macrophages are polarized by Th 2-derived cytokines, promoting tissue repair. Meanwhile, M2-like macrophages can support angiogenesis and express immunosuppressive molecules by secreting adrenomedullin and Vascular Epithelial Growth Factor (VEGF), thereby promoting tumor growth.
Unlike CAR-T cells, CAR-M cells have the following advantages: 1) T cells cannot enter the tumor environment due to the physical barrier formed by the stroma surrounding the tumor cells, and macrophages can significantly infiltrate into the tumor environment. TAMs play an important role in tumor invasion, metastasis, immunosuppression and angiogenesis. CAR-M can reduce the proportion of TAM, affect the cell phenotype of TAM, and has positive effect on tumor treatment. 2) In addition to having the effect of phagocytizing tumor cells, CAR-M also has the effect of promoting antigen presenting ability and enhancing T cell killing. 3) Compared to CAR-T, CAR-M has a limited circulation time and less non-tumor targeting toxicity. 4) Unlike CAR-T cells, CAR-M cells have a lower risk of developing GVHD, meaning that CAR-M has the potential to be a "off-the-shelf" general purpose cytodrug.
Although CAR-M has great potential as a powerful tumor immunotherapy approach, many problems need to be overcome to achieve the desired effect. First, cell number limitation: macrophages will not proliferate either after in vitro or in vivo injection. Patients can only receive a limited number of macrophages, which may affect the therapeutic effect. The second is the migration characteristics of macrophages in vivo: exogenous macrophages pass through the lungs after injection and then leave most of them in the liver, which is detrimental to cancer treatment. Finally, attempts to attack cancer by genetically engineered cells have been kept in mind because macrophages are not easily transfected with standard viral vectors used in gene and cell therapies.
Disclosure of Invention
The application aims to provide a CAR-macrophage and a preparation method thereof, and aims to solve the technical problem of how to enhance the efficient recognition and phagocytosis of PD-L1 positive lung cancer cells by the macrophage.
In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the application provides a method of preparing a CAR-macrophage, comprising the steps of:
constructing a lentivirus containing a nucleotide sequence shown in SEQ ID No. 1;
transfection of the lentivirus to CD34 + In hematopoietic stem cells or induced pluripotent stem cells, the expressed amino acid sequence shown in SEQ ID No.2 is obtainedSeed cells of chimeric antigen receptor of (a);
and (3) carrying out induced differentiation treatment on the seed cells capable of expressing the chimeric antigen receptor in a macrophage differentiation medium to obtain the CAR-macrophages.
In a second aspect, the application also provides a CAR-macrophage prepared by the method for preparing a CAR-macrophage according to the application.
The application provides a preparation method of CAR-macrophage derived from artificial blood stem cells or induced pluripotent stem cells and the CAR-macrophage obtained by the preparation method, which comprises the steps of firstly constructing a lentivirus containing a nucleotide sequence shown as SEQ ID No.1, expressing a chimeric antigen receptor of PD-L1 molecules specifically recognizing the surface of tumor cells, and utilizing the chimeric antigen receptor to carry out the preparation on human CD34 + The hematopoietic stem cells or the induced pluripotent stem cells are subjected to genetic modification, seed cells which stably express the chimeric antigen receptor shown in SEQ ID No.2 are screened, and the seed cells which can express the chimeric antigen receptor are subjected to induced differentiation in vitro, so that CD34 can be obtained + Hematopoietic stem cells or induced pluripotent stem cell-derived CAR-macrophages. The sample application not only solves the defect that macrophages cannot proliferate, but also can greatly enhance the targeting property of the CAR-macrophages and the phagocytic capacity of tumor cells, so the CAR-macrophages obtained by the preparation method can be used for treating tumors and have good application prospect in clinic.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments or the description of the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram of a PD-L1-CAR lentiviral vector provided by an embodiment of the application;
FIG. 2 is an electrophoretogram of the enzyme digestion identification of the PD-L1-CAR lentiviral expression plasmid provided by the embodiment of the application; wherein M is a DNA Marker lane, 1 is a lane before the digestion of the PD-L1-CAR plasmid, and 2 is a lane after the digestion of the PD-L1-CAR plasmid;
FIG. 3 is a flow chart of the detection of CD34 expression on the surface of human umbilical cord blood-derived HSCs cells provided in an embodiment of the present application;
FIG. 4 shows GFP provided by an embodiment of the present application + PD-L1-CAR-HSCs cell flow assay;
FIG. 5 is a flow chart of a PD-L1-CAR-hM cell phenotype provided by an embodiment of the application; wherein a is CD14 + Flow detection of PD-L1-CAR-hM cell phenotype, b is CD11b + Flow detection of PD-L1-CAR-hM cell phenotype;
FIG. 6 shows GFP provided by an embodiment of the present application + PD-L1-CAR-iPSCs cell flow detection diagram;
FIG. 7 is a flow chart of a PD-L1-CAR-iM cell phenotype provided by an embodiment of the application; wherein a is CD14 + Flow detection of PD-L1-CAR-iM cell phenotype, b is CD11b + Flow detection of PD-L1-CAR-iM cell phenotype;
FIG. 8 is a flow chart of the lung cancer cell surface PD-L1 expression provided by the embodiment of the application; wherein a is the flow detection of lung cancer cell A549 and b is the flow detection of lung cancer cell H1299;
FIG. 9 is a flow assay of PD-L1-CAR-hM phagocytic lung cancer cells provided by an embodiment of the application; wherein a is the flow detection of lung cancer cell A549 and b is the flow detection of lung cancer cell H1299;
FIG. 10 is a flow chart of a PD-L1-CAR-iM cell phagocytic lung cancer cell provided by an embodiment of the application; wherein a is the flow detection of lung cancer cell A549 and b is the flow detection of lung cancer cell H1299.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
It should be understood that, in various embodiments of the present application, the sequence number of each process described above does not mean that the execution sequence of some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.
The embodiment of the application provides a preparation method of CAR-macrophage, which comprises the following steps:
s01: constructing a lentivirus containing a nucleotide sequence shown in SEQ ID No. 1;
s02: transfection of lentiviruses into CD34 + Obtaining seed cells capable of expressing chimeric antigen receptor with amino acid sequence shown as SEQ ID No.2 in hematopoietic stem cells or induced pluripotent stem cells;
s03: and (3) carrying out induced differentiation treatment on the seed cells capable of expressing the chimeric antigen receptor in a macrophage differentiation medium to obtain the CAR-macrophages.
The preparation method of the CAR-macrophage constructs a slow virus containing a nucleotide sequence shown in SEQ ID No.1, which can express a chimeric antigen receptor of PD-L1 molecules specifically recognizing the surface of tumor cells; specifically, it can express chimeric antigen receptor whose amino acid sequence is shown as SEQ ID No.2, i.e. the chimeric antigen receptor is a protein composed of amino acid sequence shown as SEQ ID No. 2. Wherein, amino acids 1 to 22 are Signal Peptides (SP) fragments, amino acids 23 to 284 are PD-L1 scFv antibodies, amino acids 285 to 355 are CD8 alpha domain, amino acids 356 to 416 are CD86 domain, amino acids 417 to 477 are FcGamma R I domain, and the specific chimeric antigen receptor can be well expressed in CAR-macrophages.
SEQ ID No.1：
atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccaatgaagcacctgtggttctttctgctgctggtggccgcccccagatgggtgctgtccgaggtgaagctgcaggagtcaggacctagcctggttaggcctggggcttcagtgaagatatcctgcaaggcttctgcttactcattcaccagctactggatgcactgggtgaagcagaggcctggacaaggtcttgactggattggcatgattgatccttccgacagtgaagctaggttaaatcagaagttcaaggacaaggccacattgactgtagacaaatcctccagtacaacctacattcacctcagcagcccgacatctgaggactctgcggtctattactgtgcaagatcttatggttacgacggggactactacctcgatgtctggggcgcagggaccacggtcaccgtctcctcaggcggcggcagcggcggcggctctggaggaggatctggcggcggaagcgatattgtgatgacccagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggcaagccaagacattaacaaatatatagcttggtaccaacacaagcctggaaaaggtcctaggctgctcatacagtacacatctacattacagccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccttcagcatcagcaacctggagcctgaagatattgcaacttattattgtctacagtatgattatcttcggacgttcggtggaggcaccaagctggaaatcaaagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttacaaatggaagaagaagaagcggcctcgcaactcttataaatgtggaaccaacacaatggagagggaagagagtgaacagaccaagaaaagagaaaaaatccatatacctgaaagatctgatgaagcccagcgtgtttttaaaagttcgaagacatcttcatgcgacaaaagtgatacatgttttcgtaaagaactgaaaagaaagaaaaagtgggatttagaaatctctttggattctggtcatgagaagaaggtaatttccagccttcaagaagacagacatttagaagaagagctgaaatgtcaggaacaaaaagaagaacagctgcaggaaggggtgcaccggaaggagccccagggggccacg。
SEQ ID No.2：
MLLLVTSLLLCELPHPAFLLIPMKHLWFFLLLVAAPRWVLSEVKLQESGPSLVRPGASVKISCKASAYSFTSYWMHWVKQRPGQGLDWIGMIDPSDSEARLNQKFKDKATLTVDKSSSTTYIHLSSPTSEDSAVYYCARSYGYDGDYYLDVWGAGTTVTVSSGGGSGGGSGGGSGGGSDIVMTQSPSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRLLIQYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDYLRTFGGGTKLEIKAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCFRKELKRKKKWDLEISLDSGHEKKVISSLQEDRHLEEELKCQEQKEEQLQEGVHRKEPQGAT。
The CAR-macrophage is prepared by modifying macrophage with chimeric antigen receptor, and can be CD34 + Hematopoietic stem cells or induced pluripotent stem cell-derived CAR-macrophages. When seed cells expressing the chimeric antigen receptor are obtained by transfection of lentiviruses into CD34 + When hematopoietic stem cells are obtained, human Hematopoietic Stem Cell (HSCs) -derived CAR-macrophages (denoted by PD-L1-CAR-hM) are obtained, and when seed cells capable of expressing the chimeric antigen receptor are obtained by transfecting lentiviruses into induced pluripotent stem cells, induced pluripotent stem cell (Induced pluripotent stem cells, iPSCs) -derived CAR-macrophages (denoted by PD-L1-CAR-iM) are obtained.
In one embodiment, when the seed cells are obtained by transfection of lentiviruses into CD34 + When obtained from hematopoietic stem cells, the macrophage differentiation medium for inducing differentiation of seed cells includes:
differentiation medium M1: RPMI1640 culture solution, 1.8-2.2% (v/v) B27 additive, 1.8-2.2 mM L-alanyl-L-glutamine, 0.8-1.2% (v/v) nonessential amino acid, 46-54 ng/ml vitamin C, 0.8-1.2 ng/ml human interleukin-3 and 46-54 ng/ml human macrophage colony stimulating factor;
differentiation medium M2: RPMI1640 medium, 1.8-2.2% (v/v) B27 additive, 1.8-2.2 mM L-alanyl-L-glutamine, 0.8-1.2% (v/v) nonessential amino acid, 46-54 ng/ml vitamin C and 46-54 ng/ml human macrophage colony stimulating factor.
By using the differentiation medium M1 and the differentiation medium M2 in combination, the above-mentioned methods can be usedHematopoietic stem cells are better induced to differentiate into macrophages, and the above-described RPMI1640 culture broth and related additives or cytokines are commercially available. Further, the induced differentiation treatment includes: seed cells expressing the chimeric antigen receptor are cultured in differentiation medium M1 for 4-6 days, and then in differentiation medium M2 for 2-4 days. Further, the conditions for the culture in the differentiation medium M1 and the conditions for the culture in the differentiation medium M2 include: 36.5-37.5 ℃ and 4.5-5.5 percent CO 2 . The induced differentiation effect is better under the above conditions.
In one embodiment, lentiviruses are transfected into CD34 + The steps in hematopoietic stem cells or induced pluripotent stem cells include: infection of CD34 with lentiviruses + Hematopoietic stem cells or induced pluripotent stem cells are treated with puromycin for 45-60 h and then 2-3 days. Clones that more stably express the chimeric antigen receptor can thus be selected to construct stable cell lines. Wherein the multiplicity of infection (MOI) of lentiviral infection is 45-65. The concentration of puromycin is 0.24-0.26 mug/ml. The effect of infection under the above conditions is even greater. Further, CD34 + The preparation method of the hematopoietic stem cells comprises the following steps: peripheral blood mononuclear cells isolated from human umbilical cord blood are resuspended in hematopoietic stem cell complete medium and then inoculated into cell culture flasks using CD34 + Hematopoietic stem cells are cultured in complete medium for 8-10 days. While induced pluripotent stem cells with unlimited proliferation capacity are prepared by selecting healthy human Peripheral Blood Mononuclear Cells (PBMCs).
In one embodiment, when the seed cells are obtained by transfecting lentiviruses into induced pluripotent stem cells, the step of inducing differentiation comprises: digesting and re-suspending seed cells capable of expressing chimeric antigen receptor to obtain embryoid body; placing the embryoid body in an APEL II culture medium added with 9-11 ng/ml BMP-4 and 4-6 ng/ml bFGF for first culture to obtain a first culture product; placing the first culture product in an APEL II culture medium added with 9-11 ng/ml BMP-4, 4-6 ng/ml bFGF, 46-54 ng/ml VEGF and 90-110 ng/ml SCF for second culture to obtain a second culture product; producing the second culture productPlacing the culture in an APEL II culture medium added with 9-11 ng/ml bFGF, 46-54 ng/ml VEGF, 46-54 ng/ml SCF, 9-11 ng/ml IGF-1, 22-28 ng/ml IL-3, 46-54 ng/ml M-CSF and 46-54 ng/ml GM-CSF for third culture to obtain a third culture product; the third culture product is placed in StemPro with 4-6 ng/ml bFGF, 46-54 ng/ml VEGF, 46-54 ng/ml SCF, 9-11 ng/ml IGF-1, 22-28 ng/ml IL-3, 46-54 ng/ml M-CSF and 46-54 ng/ml GM-CSF added TM -34SFM culture medium for fourth culture to obtain a fourth culture product; placing the fourth culture product into a marrow maturation medium (MM medium) containing 4-6 ng/ml bFGF, 46-54 ng/ml VEGF, 46-54 ng/ml SCF, 9-11 ng/ml IGF-1, 22-28 ng/ml IL-3, 90-110 ng/ml M-CSF and 90-110 ng/ml GM-CSF for fifth culture, and obtaining a fifth culture product; the fifth culture product was placed in a myeloid-series maturation medium containing no IL-3 for a sixth culture.
Further: the first cultivation time is 20-24 hours, the second cultivation time is 6-7 days, the third cultivation time is 1-2 days, the fourth cultivation time is 9-10 days, the fifth cultivation time is 2-3 days, and the sixth cultivation time is 5-6 days. Wherein the fourth culturing step comprises: inoculating 40-50 embryoid bodies in the third culture product into a pore plate which is coated with matrigel in advance for culture; the fifth culturing step comprises: the suspension cells in the fourth culture product were re-inoculated into a well plate previously coated with matrigel for culture. The differentiation efficiency of embryoid bodies can be promoted by coating matrigel.
In the above procedure, APEL II is a well defined, serum and animal source free medium for human iPSC cell differentiation that supports endodermal, mesodermal and ectodermal differentiation when specific cytokines or induction factors are added. StemPro TM -34SFM medium is a serum-free medium formulated to support human hematopoietic cell culture. The marrow maturation medium is called MM medium for inducing and differentiating hematopoietic progenitor cells into macrophages. The above media and related cytokines or inducers are commercially available.
Further, the method for preparing the induced pluripotent stem cell-derived CAR-macrophage further comprises the following steps: placing the CAR-macrophage obtained by induction differentiation treatment in an RPMI1640 culture medium containing 9-11% FBS for culture; or the CAR-macrophage obtained by induction differentiation treatment is placed in an H3000 culture medium containing 90-110 ng/mL M-CSF and 90-110 ng/mL GM-CSF for culture. RPMI1640 is a universal basal medium for cell culture; the H3000 medium is a xeno-free medium for hematopoietic cell expansion. The further culture described above allows for large scale expansion of the differentiated cells to obtain a large number of induced pluripotent stem cell-derived CAR-macrophages.
In one embodiment, the method of preparing a lentivirus comprises: the nucleotide sequence shown in SEQ ID No.2 is inserted into a pLVX-EF1 alpha-AcGFP 1-N1 vector to obtain a slow virus vector, the slow virus vector and packaging vectors pMD2.G and psPAX2 are transfected into 293T cells, and the packaging is carried out when the fusion degree of the 293T cells is 70% -80%.
Correspondingly, the embodiment of the application also provides the CAR-macrophage, which is prepared by the preparation method of the CAR-macrophage.
According to the embodiment of the application, the gene fragment (shown as SEQ ID No. 2) of the chimeric antigen receptor is utilized to carry out gene modification on human blood stem cells or induced pluripotent stem cells, seed cells with stable expression shown as SEQ ID No.2 are screened out, and are stored after being amplified in a large quantity, so that the defect that macrophages cannot proliferate can be overcome as seed cells of subsequent CAR-macrophages. And the seed cells are induced and differentiated in vitro to obtain human hematopoietic stem cells or induced pluripotent stem cell-derived CAR-macrophages, and the CAR-macrophages can perform efficient recognition and phagocytosis on PD-L1 positive lung cancer cells in vitro, so that the targeting of the CAR-macrophages and the recognition and phagocytosis capacity of tumor cells such as a non-small cell lung cancer cell strain H1299 can be greatly enhanced.
The following description is made with reference to specific embodiments.
Example 1 preparation of PD-L1-CAR-hM cells
1. Construction of PD-L1-CAR lentiviral vector and preparation of PD-L1-CAR lentivirus
(1) Construction of PD-L1-CAR lentiviral vector
The PD-L1-CAR sequence (SEQ ID No. 1) was designed and synthesized by early experimental screening and investigation: from the 5 'end to the 3' end are the signal peptide SP, PD-L1 scFv antibody, the transmembrane region of CD8 alpha, the intracellular functional region of CD86 and the intracellular functional region of Fcgamma R I. The synthesized PD-L1-CAR sequence was cloned into a pLVX-EF1 alpha-AcGFP 1-N1 vector to obtain a PD-L1-CAR lentiviral vector, as shown in FIG. 1.
(2) Preparation of PD-L1-CAR lentiviruses
The PD-L1-CAR lentivirus vector was prepared by co-transfecting 293T cells with the lentiviral packaging vector pMD2.G, psPAX 2. Packaging the lentivirus when the fusion degree of 293T cells is 70% -80%. Collecting virus supernatant at 24h and 48h of lentiviral package, centrifuging at 4000rpm for 10min, filtering with 0.45um filter membrane to remove cell debris, centrifuging at 25000rpm at ultrahigh speed for 2h at 4deg.C to obtain PD-L1-CAR lentiviral concentrate, packaging, and storing at-80deg.C.
(3) Identification of PD-L1-CAR lentiviral vectors
After the constructed PD-L1-CAR lentiviral expression plasmid is cut by using restriction enzymes EcoRI and BamHI, electrophoresis is carried out for 40 minutes by using 1.2% agarose gel under the voltage of 120V, and the electrophoresis band is analyzed to judge whether the recombinant PD-L1-CAR lentiviral expression plasmid is correctly inserted into the PD-L1 gene fragment, and the electrophoresis detection result is shown in figure 2.
2. Expansion culture of human umbilical cord blood-derived hematopoietic stem cells
Preparation of hematopoietic stem cell complete medium: hematopoietic cell serum-free medium StemSpan TM SFEM (Stem Cell, 09600) added cytokines SCF (100 ng/ml), flt-3L (100 ng/ml), TPO (50 ng/ml), IL-6 (50 ng/ml), LDL (10 ug/ml), SR1 (750 nM) and UM171 (35 nM).
Day 1: isolation of Peripheral Blood Mononuclear Cells (PBMCs) from umbilical cord blood, resuspension of PBMCs with formulated hematopoietic Stem cell complete Medium, cell Density 5X 10 6 Inoculating 10mL of the culture medium to a T25 cell culture flask, vertically placing the T25 cell culture flask on a shaking table, rotating the shaking table at 100rpm/min, and culturing with carbon dioxideBox, 37 ℃,5% co 2 Is cultured. Day 2-5, 2-3mL fresh CD34 are added daily + The rotation speed of the shaking table can be adjusted to 110-120rpm/min when the culture volume of each culture flask reaches 20ml-25 ml. On days 6-9, 4-6mL of fresh CD34 are added daily + Hematopoietic stem cells complete medium. Day 9 samples were counted and CD34 was detected by flow-through + Proportion of Hematopoietic Stem Cells (HSCs). The results are shown in FIG. 3, CD34 + HSCs cell fraction was 72.20%.
3. Preparation of PD-L1-CAR-HSCs cells
PD-L1-CAR lentivirus infects HSCs cells: HSCs cells were infected with PD-L1-CAR lentiviruses (MOI=60) and treated with puromycin (0.25. Mu.g/ml) for 3 days 48 hours post infection to screen out HSCs cells stably expressing PD-L1-CAR, construct PD-L1-CAR-HSCs stable cell lines, and after massive expansion, frozen in liquid nitrogen for use.
By detecting GFP + The proportion of PD-L1-CAR-HSCs cells indirectly detects the infection efficiency of PD-L1-CAR lentiviruses on HSCs cells. The results are shown in FIG. 4, GFP + The proportion of PD-L1-CAR-HSCs reached 74.16%.
4. Induction of differentiation of PD-L1-CAR-HSCs into PD-L1-CAR-hM
Preparing macrophage differentiation medium M1: RPMI1640 medium was supplemented with 2% (v/v) B27 supplement, 2mM Glutamax, 1% (v/v) nonessential amino acid (NEAA), 50ng/ml vitamin C, 10ng/ml human interleukin-3 (IL-3) and 50ng/ml human macrophage colony stimulating factor (M-CSF).
Preparing macrophage differentiation medium M2: RPMI1640 medium was supplemented with 2% (v/v) B27 additive, 2mM GlutamaxL-alanyl-L-glutamine, 1% (v/v) non-essential amino acid (NEAA), 50ng/ml vitamin C and 50ng/ml human macrophage colony stimulating factor (M-CSF).
The PD-L1-CAR-HSCs cells in step 3 were resuspended in M1 medium at 37℃in 5% CO 2 Culturing in incubator for 5 days, and changing M1 culture medium at half intervals. After 5 days, the cells were collected, centrifuged and resuspended in M2 medium at 37℃and 5% CO 2 Culturing in incubator for 3 days to obtainHuman hematopoietic stem cell-derived CAR-macrophages (PD-L1-CAR-hM).
5. Identification of PD-L1-CAR-hM cells
PD-L1-CAR-hM cells obtained by induced differentiation were collected, resuspended in PBS containing 0.1% BSA, and after incubation with CD14-PE (399202, biolegend) and CD11b-FITC (301330, biolegend) antibodies for 20 minutes at room temperature in the absence of light, the expression of CD14 and CD11b on the surface of the PD-L1-CAR-hM cells was detected and analyzed by flow cytometry.
The expression of CD14 and CD11b on the surface of PD-L1-CAR-hM cells derived from human hematopoietic stem cells was examined by flow cytometry, and the results are shown in FIG. 5, CD14 + PD-L1-CAR-hM cell ratio of 99.98%, CD11b + The proportion of PD-L1-CAR-hM cells was 99.97%. We demonstrate that we successfully obtained human HSCs-derived CAR-macrophages by inducing differentiation.
Example 2 preparation of PD-L1-CAR-iM cells
1. Construction of PD-L1-CAR lentiviral vector and preparation of PD-L1-CAR lentivirus
The same as in example 1.
2. Preparation of PD-L1-CAR-iPSCs cells
PD-L1-CAR lentivirus infected iPSCs cells: healthy volunteers, PBMCs from which the laboratory has been successfully reprogrammed, were selected, and after resuscitating and expansion culture, iPSCs cells (MOI=60) were infected with PD-L1-CAR lentiviruses and treated with puromycin (0.25. Mu.g/ml) for 3 days 48 hours after infection to screen iPSCs clones stably expressing PD-L1-CAR, and a PD-L1-CAR-iPSCs stable cell line was constructed, and after a large expansion, frozen in liquid nitrogen for use.
By detecting GFP + The ratio of PD-L1-CAR-iPSCs is indirectly detected, and the infection efficiency of PD-L1-CAR lentivirus to iPSCs is indirectly detected. The results are shown in FIG. 6, GFP + The proportion of PD-L1-CAR-iPSCs cells reached 87.74%.
3. Induction of differentiation of PD-L1-CAR-iPSCs into PD-L1-CAR-iM
Day 0: undifferentiated PD-L1-CAR-iPSCs were digested with ReLeSR (05872,STEMCELL Technologies) as a digestive juice and then cultured with mTESR as a human-inducible pluripotent stem cell culture medium85850,STEMCELL Technologies) are resuspended and transferred to an ultra-low adsorption plate (3471, corning) at 37℃with 5% CO 2 Culturing overnight under 50rpm vibration to form Embryoid Bodies (EBs).
Day 1: the resulting EBs were cultured in APEL II medium (05270,STEMCELL Technologies) supplemented with BMP-4 (10 ng/ml) and bFGF (5 ng/ml) to give primitive streak mesodermal progenitor cells.
Day 2-7: BMP-4 (10 ng/ml), bFGF (5 ng/ml), VEGF (50 ng/ml) and SCF (100 ng/ml) were added to APEL II medium and differentiated in the hematopoietic direction;
day 8-9: bFGF (10 ng/ml), VEGF (50 ng/ml), SCF (50 ng/ml), IGF-1 (10 ng/ml), IL-3 (25 ng/ml), M-CSF (50 ng/ml) and GM-CSF (50 ng/ml) were added to APEL II medium to achieve differentiation in the myeloid lineage;
day 10: 40-50 EBs after 9 days differentiation were inoculated into 1 well of 6-well plate pre-coated with Matrigel matrix (354277, corning) with medium exchange for Stempro containing bFGF (5 ng/ml), VEGF (50 ng/ml), SCF (50 ng/ml), IGF-1 (10 ng/ml), IL-3 (25 ng/ml), M-CSF (50 ng/ml) and GM-CSF (50 ng/ml) TM -34SFM medium (10639011, gibco);
on day 20, suspension cells in wells were harvested and re-inoculated into fresh Matrigel matrix pre-coated wells and cultured in myeloid maturation medium (MM medium) containing bFGF (5 ng/ml), VEGF (50 ng/ml), SCF (50 ng/ml), IGF-1 (10 ng/ml), IL-3 (25 ng/ml), M-CSF (100 ng/ml) and GM-CSF (100 ng/ml) for 2 days;
culturing cells on days 22-27 with a myeloid maturation medium that does not contain IL-3;
from day 28, successfully differentiated iPSCs-derived CAR-macrophages (PD-L1-CAR-iM) were cultured in RPMI1640 medium containing 10% FBS or H3000 medium containing M-CSF (100 ng/mL) and GM-CSF (100 ng/mL).
4. Identification of PD-L1-CAR-iM cells
PD-L1-CAR-iM cells after 28 days of induced differentiation were collected, resuspended in PBS containing 0.1% BSA, and after incubation with CD14-PE (399202, biolegend) and CD11b-FITC (301330, biolegend) antibodies for 20 minutes at room temperature in the absence of light, the expression of CD14 and CD11b on the surface of the PD-L1-CAR-iM cells was detected and analyzed by flow cytometry.
The results are shown in FIG. 7, CD14 + PD-L1-CAR-iM cell ratio of 99.95%, CD11b + The proportion of PD-L1-CAR-iM cells was 99.92%, which demonstrates that we successfully obtained human iPSCs-derived CAR-macrophages by induction differentiation.
EXAMPLE 3 phagocytosis assay of lung cancer cells in vitro
(1) Detection of PD-L1 expression on surfaces of lung cancer cells A549 and H1299
Lung cancer cells a549 and H1299 were collected, incubated with PD-L1-FITC antibody (393605, biolegend) at room temperature for 20 minutes in the absence of light, and expression of PD-L1 on the cell surfaces of a549 and H1299 was detected and analyzed by flow cytometry. The result is shown in FIG. 8, PD-L1 + A549 cells at a ratio of 7.56%, PD-L1 + The proportion of H1299 cells was 86.18%. The results show that: the surface of the lung cancer cell A549 is low in expression of the PD-L1 molecule, and the surface of the lung cancer cell H1299 is high in expression of the PD-L1 molecule.
(2) Phagocytic function detection of CAR-macrophages on lung cancer cells in vitro
Lung cancer cells a549 and H1299 were co-cultured with CellTracker dark red dye (C34565, thermo Fisher) at a ratio of 1:5 (cell number ratio) with CellTracker CMFDA dye (C7025, thermo Fisher) labeled PD-L1-CAR-hM cells of example 1 (or PD-L1-CAR-iM cells of example 2). After 24 hours, cells were washed with PBS, treated with trypsin, and phagocytic function of PD-L1-CAR-hM and PD-L1-CAR-iM on lung cancer cells was detected and analyzed with a flow cytometer.
The results are shown in fig. 9 and 10: the phagocytosis ratio of PD-L1-CAR-hM cells to PD-L1 low-expression A549 cells is 55.60 percent, and the phagocytosis ratio to PD-L1 high-expression H1299 cells is 78.17 percent; the phagocytosis ratio of PD-L1-CAR-iM cells to PD-L1 low-expression A549 cells is 52.41%, and the phagocytosis ratio to PD-L1 high-expression H1299 cells is 73.22%. The results prove that the CAR-macrophages derived from the induced pluripotent stem cells or the CAR-macrophages derived from the artificial blood stem cells obtained by the embodiment of the application have stronger and more specific phagocytic functions on PD-L1 positive lung cancer cells.
The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.
Sequence listing
<110> Beijing Sier Biotech Limited liability company
<120> CAR-macrophages and methods of making the same
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1431
<212> DNA
<213> Artificial Sequence
<400> 1
atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60
atcccaatga agcacctgtg gttctttctg ctgctggtgg ccgcccccag atgggtgctg 120
tccgaggtga agctgcagga gtcaggacct agcctggtta ggcctggggc ttcagtgaag 180
atatcctgca aggcttctgc ttactcattc accagctact ggatgcactg ggtgaagcag 240
aggcctggac aaggtcttga ctggattggc atgattgatc cttccgacag tgaagctagg 300
ttaaatcaga agttcaagga caaggccaca ttgactgtag acaaatcctc cagtacaacc 360
tacattcacc tcagcagccc gacatctgag gactctgcgg tctattactg tgcaagatct 420
tatggttacg acggggacta ctacctcgat gtctggggcg cagggaccac ggtcaccgtc 480
tcctcaggcg gcggcagcgg cggcggctct ggaggaggat ctggcggcgg aagcgatatt 540
gtgatgaccc agtctccatc ctcactgtct gcatctctgg gaggcaaagt caccatcact 600
tgcaaggcaa gccaagacat taacaaatat atagcttggt accaacacaa gcctggaaaa 660
ggtcctaggc tgctcataca gtacacatct acattacagc caggcatccc atcaaggttc 720
agtggaagtg ggtctgggag agattattcc ttcagcatca gcaacctgga gcctgaagat 780
attgcaactt attattgtct acagtatgat tatcttcgga cgttcggtgg aggcaccaag 840
ctggaaatca aagcgaagcc caccacgacg ccagcgccgc gaccaccaac accggcgccc 900
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 960
gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 1020
gggacttgtg gggtccttct cctgtcactg gttatcaccc tttacaaatg gaagaagaag 1080
aagcggcctc gcaactctta taaatgtgga accaacacaa tggagaggga agagagtgaa 1140
cagaccaaga aaagagaaaa aatccatata cctgaaagat ctgatgaagc ccagcgtgtt 1200
tttaaaagtt cgaagacatc ttcatgcgac aaaagtgata catgttttcg taaagaactg 1260
aaaagaaaga aaaagtggga tttagaaatc tctttggatt ctggtcatga gaagaaggta 1320
atttccagcc ttcaagaaga cagacattta gaagaagagc tgaaatgtca ggaacaaaaa 1380
gaagaacagc tgcaggaagg ggtgcaccgg aaggagcccc agggggccac g 1431
<210> 2
<211> 477
<212> PRT
<213> Artificial Sequence
<400> 2
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Met Lys His Leu Trp Phe Phe Leu Leu Leu
20 25 30
Val Ala Ala Pro Arg Trp Val Leu Ser Glu Val Lys Leu Gln Glu Ser
35 40 45
Gly Pro Ser Leu Val Arg Pro Gly Ala Ser Val Lys Ile Ser Cys Lys
50 55 60
Ala Ser Ala Tyr Ser Phe Thr Ser Tyr Trp Met His Trp Val Lys Gln
65 70 75 80
Arg Pro Gly Gln Gly Leu Asp Trp Ile Gly Met Ile Asp Pro Ser Asp
85 90 95
Ser Glu Ala Arg Leu Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr
100 105 110
Val Asp Lys Ser Ser Ser Thr Thr Tyr Ile His Leu Ser Ser Pro Thr
115 120 125
Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Tyr Gly Tyr Asp
130 135 140
Gly Asp Tyr Tyr Leu Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val
145 150 155 160
Ser Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
165 170 175
Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
180 185 190
Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn
195 200 205
Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu
210 215 220
Leu Ile Gln Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe
225 230 235 240
Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu
245 250 255
Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Tyr Leu
260 265 270
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ala Lys Pro Thr
275 280 285
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
290 295 300
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
305 310 315 320
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
325 330 335
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
340 345 350
Thr Leu Tyr Lys Trp Lys Lys Lys Lys Arg Pro Arg Asn Ser Tyr Lys
355 360 365
Cys Gly Thr Asn Thr Met Glu Arg Glu Glu Ser Glu Gln Thr Lys Lys
370 375 380
Arg Glu Lys Ile His Ile Pro Glu Arg Ser Asp Glu Ala Gln Arg Val
385 390 395 400
Phe Lys Ser Ser Lys Thr Ser Ser Cys Asp Lys Ser Asp Thr Cys Phe
405 410 415
Arg Lys Glu Leu Lys Arg Lys Lys Lys Trp Asp Leu Glu Ile Ser Leu
420 425 430
Asp Ser Gly His Glu Lys Lys Val Ile Ser Ser Leu Gln Glu Asp Arg
435 440 445
His Leu Glu Glu Glu Leu Lys Cys Gln Glu Gln Lys Glu Glu Gln Leu
450 455 460
Gln Glu Gly Val His Arg Lys Glu Pro Gln Gly Ala Thr
465 470 475

Claims (10)

1. A method of preparing a CAR-macrophage, comprising the steps of:
constructing a lentivirus containing a nucleotide sequence shown in SEQ ID No. 1;
transfection of the lentivirus to CD34 + Obtaining seed cells capable of expressing chimeric antigen receptor with amino acid sequence shown as SEQ ID No.2 in hematopoietic stem cells or induced pluripotent stem cells;
and (3) carrying out induced differentiation treatment on the seed cells capable of expressing the chimeric antigen receptor in a macrophage differentiation medium to obtain the CAR-macrophages.
2. The method of claim 1, wherein when said seed cells are obtained by transfecting said lentivirus to CD34 + When obtained from hematopoietic stem cells, the macrophage differentiation medium comprises:
differentiation medium M1: RPMI1640 culture solution, 1.8-2.2% (v/v) B27 additive, 1.8-2.2 mM L-alanyl-L-glutamine, 0.8-1.2% (v/v) nonessential amino acid, 46-54 ng/ml vitamin C, 0.8-1.2 ng/ml human interleukin-3 and 46-54 ng/ml human macrophage colony stimulating factor;
differentiation medium M2: RPMI1640 medium, 1.8-2.2% (v/v) B27 additive, 1.8-2.2 mM L-alanyl-L-glutamine, 0.8-1.2% (v/v) nonessential amino acid, 46-54 ng/ml vitamin C and 46-54 ng/ml human macrophage colony stimulating factor.
3. The method of claim 2, wherein the induced differentiation treatment comprises: seed cells expressing the chimeric antigen receptor are cultured in the differentiation medium M1 for 4 to 6 days and then in the differentiation medium M2 for 2 to 4 days.
4. The method of claim 3, wherein the conditions under which the seed cells are cultured in the differentiation medium M1 and the conditions under which the seed cells are cultured in the differentiation medium M2 include: 36.5-37.5 ℃ and 4.5-5.5 percent CO 2 。
5. The method of claim 1, wherein when the seed cells are obtained by transfecting the lentivirus into an induced pluripotent stem cell, the step of inducing differentiation comprises:
digesting and resuspending seed cells capable of expressing the chimeric antigen receptor to obtain embryoid bodies;
placing the embryoid body in an APEL II culture medium added with 9-11 ng/ml BMP-4 and 4-6 ng/ml bFGF for first culture to obtain a first culture product;
placing the first culture product in an APEL II culture medium added with 9-11 ng/ml BMP-4, 4-6 ng/ml bFGF, 46-54 ng/ml VEGF and 90-110 ng/ml SCF for second culture to obtain a second culture product;
placing the second culture product in an APEL II culture medium added with 9-11 ng/ml bFGF, 46-54 ng/ml VEGF, 46-54 ng/ml SCF, 9-11 ng/ml IGF-1, 22-28 ng/ml IL-3, 46-54 ng/ml M-CSF and 46-54 ng/ml GM-CSF for third culture, and obtaining a third culture product;
placing the third culture product in a medium containing 4-6 ng/ml bFGF and 46-54 ng/ml VEGF, 46-54 ng/ml SCF, 9-11 ng/ml IGF-1, 22-28 ng/ml IL-3, 46-54 ng/ml M-CSF and 46-54 ng/ml GM-CSF StemPro TM -34SFM culture medium for fourth culture to obtain a fourth culture product;
placing the fourth culture product into a marrow maturation culture medium containing 4-6 ng/ml bFGF, 46-54 ng/ml VEGF, 46-54 ng/ml SCF, 9-11 ng/ml IGF-1, 22-28 ng/ml IL-3, 90-110 ng/ml M-CSF and 90-110 ng/ml GM-CSF for fifth culture, and obtaining a fifth culture product;
the fifth culture product is placed in a myeloid maturation medium without IL-3 for a sixth culture.
6. The method according to claim 5, wherein the first cultivation is performed for 20 to 24 hours, the second cultivation is performed for 6 to 7 days, the third cultivation is performed for 1 to 2 days, the fourth cultivation is performed for 9 to 10 days, the fifth cultivation is performed for 2 to 3 days, and the sixth cultivation is performed for 5 to 6 days.
7. The method of claim 5, wherein the fourth culturing step comprises: inoculating 40-50 embryoid bodies in the third culture product into a pore plate which is pre-coated with matrigel for culture; the fifth culturing step includes: and re-inoculating the suspension cells in the fourth culture product into a pore plate which is pre-coated with matrigel for culture.
8. The method of manufacturing according to claim 5, further comprising: placing the CAR-macrophage obtained by induction differentiation treatment in an RPMI1640 culture medium containing 9-11% FBS for culture; or the CAR-macrophage obtained by induction differentiation treatment is placed in an H3000 culture medium containing 90-110 ng/mL M-CSF and 90-110 ng/mL GM-CSF for culture.
9. The method of any one of claims 1-8, wherein the method of producing a lentivirus comprises: inserting the nucleotide sequence shown in SEQ ID No.2 into a pLVX-EF1 alpha-AcGFP 1-N1 vector to obtain a slow virus vector, co-transfecting 293T cells with packaging vectors pMD2.G and psPAX2, and packaging when the fusion degree of the 293T cells is 70% -80%.
10. A CAR-macrophage prepared by the method of any one of claims 1-9.
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