Preparation method and application of engineered immune cell and adenovirus

Abstract

The invention belongs to the field of biotechnology, viral vectors and cell therapy, and particularly relates to a preparation method and application of engineered immune cells and adenoviruses. An Ad35 fiber region of an Ad5F35 sequence is replaced by an Ad50 fiber region to construct the Ad5F50 type adenovirus, the Ad5F50 type adenovirus is used for infecting immune cells, especially macrophages, so that the infection efficiency is improved, the infection time is shortened, the macrophages are modified through a chimeric receptor CAR, the phagocytic function of the macrophages is redirected, and the targeted anti-tumor treatment effect is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, virus vector and cell therapy, and in particular relates to a preparation method of engineered immune cells and adenovirus and application thereof. Background Art

[0002] Adenovirus is a non-enveloped double-stranded DNA virus that can cause respiratory, ocular, gastrointestinal, and urinary tract infections. People with normal immune function usually present with self-limiting diseases after infection. Adults in many parts of the world have a high level of adenovirus antibody positivity. So far, more than 100 types of human adenovirus have been isolated and identified, divided into 7 subgroups (AG). It can be used as a gene delivery vector for gene therapy, cell therapy, and vaccine development. Except for group B adenovirus, the remaining groups of adenovirus use Coxsackievirus and adenovirus receptor (CAR) as their main attachment receptors; the main attachment receptor of group B adenovirus is CD46, which exists on macrophages, while CAR does not exist on macrophages.

[0003] Modifying macrophages with chimeric receptor CAR redirects their phagocytic function and leads to targeted anti-tumor therapeutic effects, which has the potential to stimulate adaptive immune responses. However, adoptive transplantation of macrophages has the following problems: 1. M1 macrophages with cellular immune function are easily polarized to the M2 state under the influence of the tumor microenvironment, and then lose their immune function and promote the occurrence and development of tumors; 2. The traditional method of patient autologous immune cell transfusion therapy faces problems such as few cell sources, low separation efficiency, high difficulty in transformation, time-consuming and labor-intensive, and primary macrophages are difficult to be genetically engineered through lentiviral infection.

[0004] The Ad5 / F35 chimeric vector (Ad5 / F35) used in the prior art is to embed the short-axis Ad35 (family B adenovirus) fiber into the Ad5 (family C adenovirus) capsid. This substitution is sufficient to transplant all the infectious properties of Ad35 to the chimeric vector. Therefore, it is urgent to study an efficient method for preparing macrophages. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide an Ad5F50 adenovirus, a construction method and an application. The present invention replaces the Ad35 fiber region of the Ad5F35 sequence with the fiber region of Ad50 to construct an Ad5F50 adenovirus, which is used to infect immune cells, especially macrophages, to improve the infection efficiency and shorten the infection time, and to modify macrophages by chimeric receptor CAR to redirect their phagocytic function and improve the targeted anti-tumor treatment effect.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions.

[0007] A method for preparing an engineered cell, comprising: Constructing an expression vector of the engineered cell based on Ad5F50 adenovirus; transfecting host cells with the expression vector of the engineered cells; amplifying the host cells to obtain a large amount of virus; The virus is used to transfect target cells to obtain engineered cells.

[0008] An Ad5F50 adenovirus, wherein the Ad35 fiber region in the Ad5F35 adenovirus structure is replaced by the Ad50 fiber region.

[0009] The method for constructing the Ad5F50 adenovirus specifically includes the following steps.

[0010] Step 1: construct Ad5F50 plasmid.

[0011] Step 2: After the Ad5F50 plasmid is cultured in a liquid shaker, the endotoxin-free plasmid is extracted in a small dose.

[0012] Step 3: The Ad5F50 plasmid was linearized by PacI digestion, and the linearized plasmid was verified by agarose gel electrophoresis to verify whether the digestion was complete and whether the plasmid was correct.

[0013] Step 4: Recover the remaining linearized plasmid by ethanol precipitation, dissolve the DNA precipitate with deionized water, and determine the concentration.

[0014] Step 5: Packaging the Ad5F50 adenovirus to obtain the Ad5F50 adenovirus.

[0015] More specifically, the Ad5F50 adenovirus packaging is to use host cells to package the adenovirus, and to use host cells to amplify the Ad5F50 adenovirus.

[0016] More specifically, the host cell is a 293T or 293A cell.

[0017] The present application also discloses a host cell, which is a 293A cell and further comprises an expression vector of Ad5F50 adenovirus.

[0018] The present application also discloses an expression vector, wherein the expression vector is an expression vector of the Ad5F50 adenovirus constructed by the method.

[0019] The use of the Ad5F50 adenovirus or the Ad5F50 adenovirus prepared by the construction method in preparing drugs for treating tumors.

[0020] More specifically, the Ad5F50 adenovirus or the Ad5F50 adenovirus prepared by the construction method is used for transfecting cells.

[0021] Furthermore, the tumor treatment drugs include gene therapy drugs, cell therapy drugs and vaccines.

[0022] The cell therapy drug is a chimeric antigen receptor macrophage CAR-M drug.

[0023] Furthermore, the macrophages include macrophages derived from induced differentiation of human induced pluripotent stem cells, macrophages derived from bone marrow, or macrophages differentiated from human peripheral blood monocytes.

[0024] An expression vector, wherein the expression vector is derived from an adenovirus plasmid.

[0025] A virus comprising the expression vector.

[0026] A method for preparing engineered cells, comprising: The expression vector of the engineered cell is constructed based on Ad5F50 adenovirus, the expression vector of the engineered cell is used to transfect host cells, the host cells are amplified to obtain a large amount of virus, and the virus is used to transfect target cells to obtain engineered cells.

[0027] Furthermore, the host cell is a 293A cell.

[0028] Furthermore, the gene and protein sequence of the expression vector are as shown in SEQ ID NO:1.

[0029] A modified chimeric antigen receptor macrophage, characterized in that it contains an agent selected from the following: nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, carbohydrates or analogs, lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof to modify the chimeric antigen receptor macrophage.

[0030] A pharmaceutical composition, characterized in that it comprises the chimeric antigen receptor macrophage or the modified chimeric antigen receptor macrophage and a pharmaceutically acceptable carrier.

[0031] Use of the chimeric antigen receptor expressed in macrophages, the genetically modified macrophages or the modified chimeric antigen receptor macrophages or the pharmaceutical composition in preparing drugs for treating tumors or cancers.

[0032] A method for treating a disease or condition associated with a tumor or cancer in a subject, comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor macrophage or the modified chimeric antigen receptor macrophage or the pharmaceutical composition.

[0033] A method for immune response to target tumor cells or tumor tissues, comprising the chimeric antigen receptor macrophage or the modified chimeric antigen receptor macrophage or the pharmaceutical composition.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0035] The present invention replaces the Ad35 fiber region of the Ad5F35 sequence with the fiber region of Ad50 to construct an Ad5F50 adenovirus, which is used to infect immune cells, especially macrophages, to improve the infection efficiency and shorten the virus infection time, and modifies macrophages through a chimeric receptor CAR to redirect their phagocytic function and improve the targeted anti-tumor treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a diagram of the structure of plasmid pAd5-Ad50-44P.

[0037] Figure 2 This is a diagram of the structure of plasmid pAd5-Ad35-44P.

[0038] Figure 3 This is a diagram of the enzyme cleavage effect detected by agarose gel electrophoresis of the linearized Ad5F50 plasmid.

[0039] Figure 4 This is a diagram of the enzyme cutting effect detected by agarose gel electrophoresis of the linearized Ad5F35 plasmid.

[0040] Figure 5 is a linear sequence diagram of Ad5F50 disclosed in the present application.

[0041] Figure 6 This is a comparison chart of the transfection efficiency of Ad5F50 and Ad5F35 disclosed in this application. Implementation

[0042] The present invention is specifically described below in conjunction with the accompanying drawings and Examples. The following examples are convenient for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged. All data were statistically analyzed to calculate the mean value and standard deviation. The significance of the data was analyzed using the statistical software Prism5.0. The intergroup comparison analysis was tested using ANOVA. P values ​​less than 0.05 were considered to have significant differences.

[0043] In order to improve the efficiency of target gene delivery, the present application provides an Ad5F50 adenovirus, which replaces the Ad35 fiber region in the Ad5F35 adenovirus structure with the Ad50 fiber region.

[0044] Specifically, the method for constructing the Ad5F50 adenovirus comprises the following steps: Step 1: construct Ad5F50 plasmid.

[0045] Step 2: After the Ad5F50 plasmid is cultured in a liquid shaker, the endotoxin-free plasmid is extracted in a small dose.

[0046] Step 3: The Ad5F50 plasmid was linearized by PacI digestion, and the linearized plasmid was verified by agarose gel electrophoresis to verify whether the digestion was complete and whether the plasmid was correct.

[0047] Step 4: Recover the remaining linearized plasmid by ethanol precipitation, dissolve the DNA precipitate with deionized water, and determine the concentration.

[0048] Step 5: Packaging the Ad5F50 adenovirus to obtain the Ad5F50 adenovirus.

[0049] like Figure 5 Shown is a linear sequence diagram of Ad5F50.

[0050] More specifically, the Ad5F50 adenovirus packaging is specifically packaging adenovirus using host cells; More specifically, a large amount of Ad5F50 adenovirus can be obtained by amplifying the Ad5F50 adenovirus using host cells.

[0051] More specifically, the host cells are mainly 293A cells, but include but are not limited to the 293A cells.

[0052] The present application also discloses an expression vector, wherein the expression vector is an expression vector of the Ad5F50 adenovirus constructed by the method.

[0053] The method comprises the step of purifying the harvested virus stock solution.

[0054] The use of the Ad5F50 adenovirus or the Ad5F50 adenovirus prepared by the construction method in preparing drugs for treating tumors.

[0055] Furthermore, the tumor treatment drugs include gene therapy drugs, cell therapy drugs and vaccines.

[0056] Furthermore, the target cell is an immune cell. More specifically, the immune cell is a macrophage.

[0057] The cell therapy drug is a chimeric antigen receptor macrophage CAR-M drug. By using the Ad5F50 adenovirus disclosed in the present application to deliver the genetic material of the chimeric antigen receptor, the research and development and production processes can be shortened. For patients, especially in terms of suppressing the condition of tumor patients using cell therapy, the treatment cycle can be effectively shortened.

[0058] Furthermore, the macrophages include macrophages derived from induced differentiation of human induced pluripotent stem cells, macrophages derived from bone marrow, or macrophages differentiated from human peripheral blood monocytes.

[0059] More specifically, the Ad5F50 adenovirus or the Ad5F50 adenovirus prepared by the construction method is used to improve the efficiency of cell transfection.

[0060] Furthermore, the cells are dividing cells and dormant cells of various mammals (especially humans).

[0061] The present application also discloses an expression vector, wherein the expression vector is derived from an adenovirus plasmid.

[0062] The present application also discloses a virus, wherein the virus comprises the expression vector.

[0063] The present application also discloses a method for preparing engineered cells, comprising: Constructing an expression vector of the engineered cell based on Ad5F50 adenovirus; The expression vector of the engineered cell is used to transfect a host cell, the host cell is amplified to obtain a large amount of virus, and the virus is used to transfect a target cell to obtain an engineered cell.

[0064] More specifically, the engineered cells can be applied to the treatment of tumors and enhance human immunity.

[0065] Furthermore, the host cell is a 293A cell.

[0066] A modified chimeric antigen receptor macrophage, characterized in that it contains an agent selected from the following: nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, carbohydrates or analogs, lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof to modify the chimeric antigen receptor macrophage.

[0067] The present application also discloses a pharmaceutical composition, characterized in that it comprises the chimeric antigen receptor macrophage or the modified chimeric antigen receptor macrophage and a pharmaceutically acceptable carrier.

[0068] More specifically, the modified chimeric antigen receptor macrophages are prepared from the Ad5F50 adenovirus.

[0069] Use of the chimeric antigen receptor expressed in macrophages, the genetically modified macrophages or the modified chimeric antigen receptor macrophages or the pharmaceutical composition in preparing drugs for treating tumors or cancers.

[0070] A method for treating a disease or condition associated with a tumor or cancer in a subject, comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor macrophage or the modified chimeric antigen receptor macrophage or the pharmaceutical composition.

[0071] A method for immune response to target tumor cells or tumor tissues, comprising the chimeric antigen receptor macrophage or the modified chimeric antigen receptor macrophage or the pharmaceutical composition.

[0072] Preparation of adenovirus type Ad5F50.

[0073] 1. Construction of plasmid.

[0074] 1. Construction of Ad5F50 plasmid.

[0075] Plasmid pAd5-Ad50-44P is a circular plasmid with a plasmid structure such as Figure 1 Said, from upstream to downstream, it is composed of the following three segments: the segment shown in the sequence of the sequence list, the genomic DNA of the recombinant adenovirus and the segment shown in the sequence of the sequence list, and further, said.

[0076] Plasmid construction methods.

[0077] 2. Construction of Ad5F35 plasmid.

[0078] Plasmid pAd5-Ad35-44P is a circular plasmid with a structure such as Figure 2The above-mentioned three segments are composed of the following three segments from upstream to downstream: the segment shown in sequence 1 of the sequence list, the genomic DNA of the recombinant adenovirus and the segment shown in sequence 2 of the sequence list.

[0079] 2. Adenovirus preparation.

[0080] 1. Preparation of Ad5F50 adenovirus.

[0081] Step 1: After overnight shaking of Ad5F50 plasmid, the endotoxin-free plasmid was extracted in small doses.

[0082] Step 2: Take 10 μg of Ad5F50 plasmid and linearize it with PacI.

[0083] Step 3: Take 5 μl of the linearized plasmid and perform agarose gel electrophoresis to verify whether the enzyme digestion is complete and whether the plasmid is correct. Figure 3 As shown, 34000 bp and 2000 bp fragments were obtained, proving that the enzyme digestion was successful.

[0084] Step 4: Recover the remaining linearized plasmid by ethanol precipitation, and dissolve the DNA precipitate in 100 μl of deionized water, with a concentration of 43.556 ng / μl.

[0085] Step 5, Ad5F50 adenovirus packaging: Use 293A cells to package adenovirus.

[0086] 2. Preparation of control virus-Ad5F35 adenovirus.

[0087] Step 1: After overnight shaking of Ad5F35 plasmid, the endotoxin-free plasmid was extracted in small doses.

[0088] Step 2: Take 10 μg of Ad5F35 plasmid and linearize it with PacI.

[0089] Step 3: Take 5 μl of the linearized plasmid and perform agarose gel electrophoresis to verify whether the enzyme digestion is complete and whether the plasmid is correct. Figure 4 As shown, 34000 bp and 2000 bp fragments were obtained, proving that the restriction enzyme digestion was successful.

[0090] Step 4: Recover the remaining linearized plasmid by ethanol precipitation, and dissolve the DNA precipitate in 100 μl of deionized water, with a concentration of 40.818 ng / μL.

[0091] Step 5: Ad5F35 adenovirus packaging: Use 293A cells to package adenovirus.

[0092] The PacI enzyme digestion reaction system is shown in the following table.

[0093]

[0094] The ethanol precipitation recovery steps are as follows.

[0095] Step 1. Add 5 μL of sodium acetate (3 M, pH 5.2) to the linearization solution and mix well to make the final concentration of sodium acetate 0.3 M.

[0096] Step 2: Add 2.5 times the volume of pre-cooled anhydrous ethanol, mix well, and place at -20 °C for 1 h.

[0097] Step 3: Centrifuge at 12000 rpm for 20 min and carefully remove the supernatant.

[0098] Step 4: Add 1 mL of pre-cooled 70% ethanol, centrifuge at 12,000 rpm for 5 min, and carefully remove the supernatant.

[0099] Step 5: Open the EP tube and place it in a biosafety cabinet to allow the remaining liquid to evaporate completely.

[0100] Step 6. Add 30 μL of deionized water to dissolve the DNA precipitate.

[0101] Virus identification and morphological observation comparison.

[0102] Comparison of cell immunofluorescence experiments.

[0103] Large-scale amplification, purification and application of adenovirus.

[0104] 1. Large-scale amplification of adenovirus.

[0105] 2. Purification of adenovirus.

[0106] 3. Titer determination.

[0107] 4. Toxicity test.

[0108] 5. Transfection efficiency: like Figure 6 The experimental data shown are the results of the comparison of the efficiency of Ad5F50 and Ad5F35 in infecting HMDM cells. Figure 1-Figure 2 These are the plasmid maps of Ad5F50 and Ad5F35, Figure 3-Figure 4 The WB results of Ad5F50 and Ad5F35 are shown respectively. Figure 6 The results show that the infection efficiency of Ad5F35 disclosed in the present application is 65.7%, while the infection efficiency of Ad5F50 is 71.7%. Based on the above experimental data, it can be seen that the infection efficiency of Ad5F50 is better than that of conventional Ad5F35.

[0109] The present application can realize the rapid preparation of engineered immune cells, especially when multiple and large-scale cell preparations are required. The use of the adenovirus disclosed in the present application can greatly reduce the preparation time of engineered cells.

[0110] It can be seen that the Ad5F50 adenovirus disclosed in this application has the following beneficial effects compared with the prior art: The present invention replaces the Ad35 fiber region of the Ad5F35 sequence with the fiber region of Ad50 to construct an Ad5F50 adenovirus, which is used to infect immune cells, especially macrophages, to improve infection efficiency and shorten virus infection time, shorten the time of target gene delivery, shorten the cycle of various cell drug research and development and production, and enable the chimeric receptor CAR to quickly modify macrophages to redirect their phagocytic function and improve the targeted anti-tumor treatment effect. Using the adenovirus disclosed in the present application, more engineered immune cells can be obtained per unit cell amount, which can save costs, reduce the production cost of immune cell drugs, and improve the therapeutic effect of unit immune cells.

Claims

1. A method for preparing engineered cells, comprising: Constructing an expression vector of the engineered cell based on Ad5F50 adenovirus; transfecting host cells with the expression vector of the engineered cells; amplifying the host cells to obtain a large amount of virus; The virus is used to transfect target cells to obtain engineered cells.

2. The method for preparing an engineered cell according to claim 1, wherein the construction of the Ad5F50 adenovirus specifically comprises the following steps: Step 1, constructing Ad5F50 plasmid; Step 2: After overnight shaking of the Ad5F50 plasmid, the endotoxin-free plasmid was extracted; Step 3, the Ad5F50 plasmid was linearized by PacI digestion, and the obtained linearized plasmid was verified by agarose gel electrophoresis to verify whether the digestion was complete and whether the plasmid was correct; Step 4: Recover the remaining linearized plasmid by ethanol precipitation, dissolve the DNA precipitate with deionized water, and measure the concentration; Step 5: Ad5F50 adenovirus packaging.

3. The method for preparing an engineered cell according to claim 1, wherein the Ad5F50 adenovirus packaging is specifically packaging adenovirus using host cells. The method for preparing engineered cells according to claim 1 , wherein the target cells are immune cells. The method for preparing engineered cells according to claim 4 , wherein the immune cells are macrophages.

6. An adenovirus, characterized in that The adenovirus is obtained by replacing the Ad35 fiber region in the Ad5F35 adenovirus structure with the Ad50 fiber region. The preparation method of the adenovirus comprises the steps of claims 1-3.

7. Use of an adenovirus in the preparation of a drug for treating tumors, wherein the adenovirus is the adenovirus according to claim 4.

8. The use of adenovirus according to claim 5 in preparing a drug for treating tumors, characterized in that: The tumor treatment drugs include gene therapy drugs, cell therapy drugs and vaccines.

9. The use of adenovirus according to claim 5 in preparing a drug for treating tumors, characterized in that: The cell therapy drug is a chimeric antigen receptor macrophage CAR-M drug.

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