Method, carrier, application and pharmaceutical composition for continuously and effectively killing macrophages

Abstract

The invention relates to the field of immune cell therapy, and provides a method for continuously and effectively killing macrophages, which is characterized by comprising the following steps: constructing a vector comprising a target gene segment, the vector being a viral vector or a non-viral vector; preparing a target immune cell based on the carrier to obtain an engineered immune cell; wherein the construction of the vector comprising the target gene segment comprises the step of coding a gene sequence of an exogenous GM-CSF protein to the vector; wherein the virus vector is an adenovirus or a lentivirus. Therefore, the preparation time of the engineered immune cells is shortened, the process is simplified, and the effect of stably killing pathogenic cells by the immune cells is achieved.

Description

Technical Field

[0001] The present invention relates to the field of immune cell therapy, and in particular to a method, a carrier, an application and a pharmaceutical composition for enabling macrophages to be continuously and effectively killed. Background Art

[0002] In the field of immune cell therapy, chimeric antigen receptor T-cell immunotherapy (CAR-T) has achieved remarkable results in the clinical treatment of hematological tumors. In the treatment of solid tumors, T cells are not a good choice for the treatment of solid tumors because the extracellular matrix (ECM) of solid tumors forms a physical barrier that hinders T cells from entering tumor tissues. Macrophages have good infiltration into tumors. At the same time, macrophages also have the functions of antigen presentation and enhancing T cell killing. Therefore, macrophages are a potential ideal immune cell for the treatment of solid tumors. Transforming macrophages into engineered cells that can target and kill cancer cells has good application prospects.

[0003] Macrophages have almost no proliferation ability. During treatment, in order to meet the need to kill tumor cells, the single injection of macrophages must reach tens of billions or even hundreds of billions. Therefore, there are certain requirements for the patient's body when collecting them. In addition, artificially modified macrophages (CAR-M) cannot proliferate. After being injected into the patient's body, they will be quickly consumed. Once the number of CAR-M is lower than a certain level, the therapeutic significance of CAR-M is lost. Therefore, providing a macrophage that can achieve self-proliferation is of great significance for the treatment of solid tumors.

[0004] In order to maintain the growth and activity of macrophages, granulocyte-macrophage colony-stimulating factor (GM-CSF) is usually added during in vitro culture. However, the artificial addition of GM-CSF in in vitro culture not only requires determining the timing and concentration of addition, but also has complicated procedures. Moreover, when macrophages are injected into the human body, they will not proliferate further. If GM-CSF and CAR-M are injected into the human body at the same time, in the complex in vivo environment, it cannot be guaranteed that the additional GM-CSF factor can fully exert its effect on macrophages. At present, a method is urgently needed to solve the above problems so that CAR-M can maintain good activity and sustained and stable killing ability. Summary of the invention

[0005] A major advantage of the present invention is that it provides a method for continuously and effectively killing macrophages, so that the prepared CAR-M always maintains an effective killing ability on target cells.

[0006] Another advantage of the present invention is that it provides a method for continuously and effectively killing macrophages, and realizes the differentiation of monocytes into macrophages through the cytokines secreted by themselves, without the need for external addition of GM-CSF cytokine.

[0007] Another advantage of the present invention is that it provides a novel adenovirus expression vector carrying GM-CSF, and immune cells modified by the adenovirus expression vector can achieve autosecretion of GM-CSF factor.

[0008] Another advantage of the present invention is that it provides a novel engineered immune cell with GM-CSF. When the engineered immune cell is a mononuclear cell, the engineered immune cell promotes itself to further differentiate into a macrophage through the GM-CSF secreted by itself. Furthermore, after the engineered immune cell differentiates into a macrophage, it still maintains the secretion of the GM-CSF factor, thereby further maintaining the growth and proliferation of the engineered macrophage itself.

[0009] Another advantage of the present invention is that it provides a novel engineered immune cell with a GM-CSF gene fragment. When the engineered immune cell is a macrophage, the engineered immune cell promotes its own proliferation through the GM-CSF factor secreted by the cell.

[0010] Another advantage of the present invention is that it provides a new type of engineered immune cell with a GM-CSF encoding gene, which can autosecrete the GM-CSF factor in the patient's body environment, thereby prolonging its maintenance time in the patient's body, further ensuring the long-term killing of target cells by immune cells with specific killing function.

[0011] Another advantage of the present invention is that it provides an application of GM-CSF engineered immune cells and a pharmaceutical composition, which is used as an immune cell drug for treating inflammatory diseases, fibrotic diseases, and solid tumor diseases, and has a sustained therapeutic effect.

[0012] Other advantages and features of the present invention will become more fully apparent from the following detailed description and will be realized and attained by the instrumentality particularly pointed out in the appended claims.

[0013] The present invention provides a method for continuously and effectively killing macrophages, characterized in that the method comprises: Constructing a vector including a target gene fragment, wherein the vector is a viral vector or a non-viral vector; Prepare target immune cells based on the vector to obtain engineered immune cells; Wherein said constructing a vector including the target gene fragment comprises encoding the gene sequence of the exogenous GM-CSF protein into said vector; The viral vector is an adenoviral vector or a lentiviral vector.

[0014] In a specific embodiment, the target gene fragment also includes a gene sequence encoding a functional chimeric receptor.

[0015] In a specific embodiment, the target immune cell is a macrophage or an immune cell capable of differentiating into a macrophage. More specifically, the immune cell capable of differentiating into a macrophage is preferably a monocyte.

[0016] According to one aspect of the present invention, a novel expression vector is provided to achieve the aforementioned objects and other objects and advantages, wherein the expression vector comprises a coding sequence encoding an exogenous GM-CSF protein.

[0017] In a specific embodiment, the expression vector is an adenovirus expression vector or a lentivirus expression vector.

[0018] The present invention also provides a method for preparing a novel expression vector with a GM-CSF coding sequence to achieve the above-mentioned and other purposes, the preparation method comprising: The target gene fragment is cloned into an adenovirus expression vector to obtain a recombinant plasmid vector, wherein the target gene fragment includes a gene sequence encoding an exogenous GM-CSF protein.

[0019] Furthermore, the gene sequence of GM-CSF is shown in SEQ ID NO:1.

[0020] More specifically, the target gene fragment also includes a gene sequence encoding a functional chimeric receptor.

[0021] Furthermore, the target immune cells may be monocytes or M (macrophage) cells.

[0022] The engineered immune cells with GM-CSF factor coding sequence prepared based on the above method not only shorten the time for preparing the engineered immune cells and simplify the process, but also stably maintain the killing effect of the engineered immune cells on target cells.

[0023] The present invention also provides the use of engineered immune cells with GM-CSF coding sequences in the preparation of immune drugs for treating inflammatory diseases, fibrotic diseases, and solid tumor diseases to achieve the above-mentioned and other purposes.

[0024] The present invention also provides an immune cell pharmaceutical composition for treating inflammatory diseases, fibrotic diseases, and solid tumor diseases to achieve the above-mentioned and other purposes.

[0025] Further objects and advantages of the present invention will be fully apparent from an understanding of the following description and the accompanying drawings.

[0026] These and other objects, features and advantages of the present invention will be more fully understood from the following detailed description, accompanying drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 FIG. 1 is a plasmid map of a GM-CSF adenovirus vector according to a preferred embodiment of the present invention.

[0028] Figure 2 The figure is a plasmid map of an adenoviral vector carrying a chimeric antigen receptor and GM-CSF according to a preferred embodiment of the present invention.

[0029] Figure 3 The data of GM-CSF secretion measured by the CAR-M capable of secreting GM-CSF factor prepared by the method of the present invention.

[0030] Figure 4 Comparison of macrophage recovery rate data calculated using GM-CSF (10 ng / mL) and M-CSF (100 ng / mL).

[0031] Figure 5A-5B According to a preferred embodiment of the present invention, human monocyte-derived macrophages were cultured in complete culture medium containing GM-CSF (10 ng / mL) and M-CSF (100 ng / mL) for 7 days, and the experimental data of macrophage activity were compared.

[0032] Figures 6A-6D The data are based on two administration methods: mouse tail vein injection and intratumoral injection, and compare the experimental data on the number change trend of CAR-M and ordinary macrophages prepared by the technical solution disclosed in this application in mice within 13 days. Implementation

[0033] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not deviate from the spirit and scope of the present invention.

[0034] The present invention provides a method for continuously and effectively killing macrophages, characterized in that the method comprises: Constructing a vector including a target gene fragment, wherein the vector is a viral vector or a non-viral vector; Prepare target immune cells based on the vector to obtain engineered immune cells; Wherein said constructing a vector including the target gene fragment comprises encoding the gene sequence of the exogenous GM-CSF protein into said vector; The viral vector is an adenoviral vector or a lentiviral vector.

[0035] In a specific embodiment, the target gene fragment also includes a gene sequence encoding a functional chimeric receptor.

[0036] In a specific embodiment, the target immune cell is an immune cell, a mononuclear cell or a macrophage that can differentiate into a macrophage. Furthermore, preferably, the target immune cell is a macrophage.

[0037] Construction of expression vector.

[0038] The construction of the expression vector is to encode the target gene into a viral vector or a non-viral vector. More specifically, the target gene at least includes a gene sequence encoding the GM-CSF factor.

[0039] Furthermore, the viral vector may be an adenoviral vector or a lentiviral vector. Furthermore, the non-viral vector may be an element that can attach to the chromosome of the target immune cell, so that the non-viral vector is replicated and passed to the offspring cells during cell division, and the expression of the target gene fragment is maintained. Based on the above expression vector disclosed in the present application, the target gene can be encoded into the target immune cell.

[0040] In a preferred embodiment, the GM-CSF expression vector is an expression vector that only carries the coding sequence of GM-CSF. Further, the plasmid map of the GM-CSF expression vector is as follows: Figure 1 shown.

[0041] More specifically, when the expression vector is an adenovirus vector, the exogenous GM-CSF protein coding sequence is packaged into an adenovirus. In a preferred embodiment, this step is to connect the gene sequence fragment encoding the exogenous GM-CSF protein to the adenovirus expression vector Ad5F35, thereby obtaining the adenovirus expression vector carrying GM-CSF.

[0042] Furthermore, the viral vector may also be a lentiviral vector.

[0043] More specifically, the promoter of GM-CSF is PGK promoter.

[0044] Compared with transfecting target immune cells with an expression vector without GM-CSF, the target immune cells prepared by transfecting target immune cells with the expression vector only carrying the GM-CSF gene fragment can secrete the GM-CSF factor by themselves, thereby simplifying the process of preparing engineered immune cells. There is no need to artificially add the GM-CSF factor during culture, and differentiation or proliferation can be achieved through the GM-CSF factor secreted by itself. In particular, when the target immune cells are macrophages, macrophages themselves cannot proliferate, but proliferation can be achieved based on the technical solution disclosed in the present application.

[0045] In another preferred embodiment, in the process of constructing the expression vector, while the GM-CSF gene fragment is cloned or inserted into the adenovirus expression vector Ad5F35, the sequence of the functional chimeric receptor is also encoded into the adenovirus expression vector Ad5F35, so that the immune cells disclosed in the present application can maintain their own growth and proliferation, and then achieve the effect of stable and continuous killing of macrophages.

[0046] More specifically, the plasmid map of the adenoviral vector carrying the functional chimeric receptor and GM-CSF coding sequences is as follows: Figure 2 shown.

[0047] More specifically, the functional chimeric receptor is a receptor that can specifically bind to a target of a pathogenic cell (target cell). More specifically, the functional chimeric receptor binds to the receptor and further transmits the signal into the cell. The functional chimeric receptor and the coding sequence of GM-CSF are added to the expression vector by gene editing at the same time, and an expression vector capable of expressing the functional exogenous receptor and the GM-CSF protein gene sequence is further obtained. More specifically, the target gene is introduced into the target immune cell through the expression vector to obtain the desired engineered immune cell that can express the functional chimeric receptor and the GM-CSF factor. By expressing the functional chimeric receptor, the engineered immune cell can achieve specific binding of the engineered immune cell to the surface antigen of the target cell, achieve killing of the target cell, and thus achieve the purpose of treating the disease. The GM-CSF factor secreted by the engineered immune cell itself maintains its own growth, differentiation or proliferation, thereby achieving the continuous and stable function of the prepared engineered immune cell.

[0048] More specifically, the functional chimeric receptor is an artificially synthesized chimeric antigen receptor or a naturally occurring receptor. Furthermore, the functional chimeric receptor is a receptor that can specifically bind to an antigen on the surface of a target cell. Furthermore, the target cell surface antigen is a protein on the surface of a pathogenic cell that causes a disease.

[0049] Furthermore, the sequence fragment of the chimeric antigen receptor can be obtained by artificial synthesis as needed.

[0050] More specifically, the viral vector is an adenovirus vector, and the adenovirus carrying the target gene fragment infects the desired target immune cells, so that the engineered immune cells that can express the functional exogenous receptor can be quickly obtained. The target immune cells are transfected with the adenovirus expression vector carrying the sequence encoding GM-CSF and the functional exogenous receptor, and the transfection efficiency is significantly improved, further shortening the preparation method of the engineered immune cells.

[0051] Furthermore, the target immune cells are monocytes or macrophages.

[0052] Furthermore, when the target immune cells are monocytes, the prepared engineered monocytes can secrete GM-CSF factors, and based on the GM-CSF factors expressed and secreted by themselves, the environment of the engineered monocytes is changed, thereby further promoting the differentiation of the engineered monocytes into engineered macrophages. Furthermore, the engineered macrophages obtained by differentiation can still express and secrete the GM-CSF factor. Based on this characteristic, the engineered macrophages can further maintain their own proliferation through the GM-CSF factor secreted by themselves.

[0053] Furthermore, in the process of infecting immune cells, the presence of GM-CSF can simplify the process of preparing engineered immune cells, saving the time and money costs of immune cell preparation. At the same time, the addition of GM-CSF can also create conditions for differentiation and proliferation for the engineered immune cells themselves by expressing and secreting the GM-CSF factor.

[0054] Example 1: A specific preparation method for sustained and stable killing engineered immune cells.

[0055] 1. A method for preparing engineered immune cells that only carry the coding sequence of GM-CSF.

[0056] (1) Connecting the GM-CSF target fragment to the adenovirus overexpression vector to obtain a recombinant plasmid vector; (2) Extraction and preparation of recombinant plasmid vectors and auxiliary plasmid vectors; (3) Virus packaging and concentration; (4) Using the packaged virus to infect target immune cells to obtain target engineered immune cells that carry the GM-CSF coding sequence.

[0057] More specifically, the target immune cells are monocytes, M (macrophage) cells, etc. Furthermore, the target immune cells are preferably macrophages.

[0058] More specifically, the target immune cells can be either autologous or allogeneic.

[0059] More specifically, the target immune cells are derived from autologous cells, which means that the target immune cells are separated and reinfused from the same patient. The target immune cells are derived from allogeneic cells, which means that the target immune cells are separated and reinfused from different people.

[0060] More specifically, the virus used in the virus packaging step is an adenovirus or a lentivirus.

[0061] Furthermore, when the target immune cells are monocytes, the engineered monocytes with GM-CSF obtained based on the above steps are affected by the GM-CSF factor, and the monocytes can be promoted to differentiate into macrophages through the GM-CSF factor secreted by themselves. Furthermore, after the monocytes differentiate into macrophages, the engineered immune cells still secrete the GM-CSF factor, thereby further promoting the growth and proliferation of the macrophages themselves, thereby ensuring the cell activity of the engineered immune cells.

[0062] Furthermore, when the target immune cells are macrophages (M) cells, the engineered M cells with GM-CSF obtained based on the above steps are affected by the GM-CSF factor, and the GM-CSF factor secreted by the engineered immune cells themselves promotes the growth and proliferation of the macrophages themselves.

[0063] Based on the engineered immune cells disclosed in the present application, the engineered immune cells can create differentiation conditions containing GM-CSF factors for themselves. The engineered monocytes and macrophages disclosed in the present application can not only promote their own differentiation and proliferation in vitro, but also promote their own differentiation and proliferation in vivo. The problem that natural monocytes themselves cannot create sufficient GM-CSF factor differentiation conditions is overcome. At the same time, the addition of GM-CSF not only promotes the differentiation, growth or proliferation of target immune cells, but also simplifies the preparation process of engineered macrophages.

[0064] In a specific embodiment, the target immune cells are mononuclear cells, further monocytes. When preparing engineered macrophages, GM-CSF factor is usually added to the culture environment. However, the engineered monocytes prepared by the present application do not need to add GM-CSF factor to prepare engineered macrophages. Therefore, the method for preparing engineered immune cells disclosed in the present application can simplify the preparation process of engineered macrophages.

[0065] Furthermore, in the above-mentioned method for preparing engineered immune cells, the step of connecting the GM-CSF target fragment with the adenovirus overexpression vector to obtain the recombinant plasmid vector is specifically as follows: The gene sequence fragment encoding the exogenous GM-CSF protein is connected to the adenovirus expression vector Ad5F35, thereby obtaining the adenovirus expression vector carrying GM-CSF.

[0066] More specifically, the promoter of GM-CSF is PGK promoter.

[0067] Furthermore, in the above-mentioned method for preparing engineered immune cells, the extraction and preparation steps of the recombinant plasmid vector and the auxiliary plasmid vector are specifically as follows: Pick a single bacterial liquid and inoculate it into Amp-resistant LB medium, and culture it overnight in a constant temperature shaker at 37°C and 200 rpm; After 16 hours, the bacterial solution was transferred to a centrifuge tube and centrifuged at 14600 rpm for 2 minutes at room temperature, and the supernatant was discarded; Add 300 μL of P1 solution and pipette more than 10 times to mix; Add 300 μL of P2 solution, mix by inverting 10 times, and then let stand for 2 minutes; Add 300 μL of P3 solution, mix by inverting 10 times, and place on ice for 10 minutes; Centrifuge at 14600 rpm for 10 minutes at room temperature and transfer the supernatant to a new centrifuge tube; Centrifuge at 14600 rpm for 5 minutes at room temperature, take 600 μL of supernatant to a new centrifuge tube, add 500 μL of isopropanol, and mix by inverting upside down twice; Centrifuge at 14600 rpm for 10 minutes at room temperature, discard the supernatant and add 1 mL of 70% ethanol; Centrifuge at 14600 rpm for 10 minutes at room temperature, discard the supernatant, and then open the lid to dry the ethanol in the tube; Finally, add 100 μL of sterile ddH2O and dissolve for 3 minutes before nucleic acid quantification.

[0068] Furthermore, in the above-mentioned method for preparing engineered immune cells, the extraction and preparation steps of the recombinant plasmid vector and the auxiliary plasmid vector also include adenovirus vector enzyme linearization, and further, the adenovirus vector enzyme linearization includes: The Ad5F35-CAR-Her2-CD3ζ-hCSF2 plasmid was linearized by restriction enzyme digestion (PacI). The restriction enzyme digestion system was as follows: 4 μl PacI, 20 μl 10× NEBuffer, 10 μg DNA, and sterile deionized water to 200 μl, and the digestion was carried out at 37°C for 8 h.

[0069] Recover the linearized plasmid by ethanol precipitation. Add 2.5 times the volume of the enzyme digestion system, pre-cooled anhydrous ethanol stored at -20℃, and mix gently up and down. Centrifuge at 12,000 rcf, 4℃ for 10 minutes, and carefully remove the supernatant. Add 1ml of pre-cooled 70% ethanol stored at -20℃, centrifuge at 12000 rcf, 4℃ for 5 minutes, carefully remove the supernatant, and aspirate all droplets on the tube wall. Place the opened EP tube in a biosafety cabinet at room temperature to allow the residual ethanol liquid to fully evaporate. Add an appropriate amount of 50μl deionized water to dissolve the DNA precipitate. After mixing, detect the linear DNA concentration.

[0070] Finally, agarose gel electrophoresis was performed to verify the correct position of the bands.

[0071] Furthermore, in the preparation method of the above-mentioned engineered immune cells, the virus packaging operation steps are specifically as follows: prepare the linearized Ad5F35 plasmid and 293A cells in advance. 293A cells were plated to a 35 mm cell culture dish 24 h before transfection, with a fusion degree of 80-90% and in good condition. The transfection plasmid is as follows: take the jetPRIME out of the 4°C refrigerator, restore it to room temperature for use, and shake it before use. Take 4 μg of the plasmid linearized after construction in Example 1, add 200 μL PRIMEBuffer, vortex for 10s; then add 8 μL PRIME, vortex for 10s, let stand at room temperature for 10min, and then add the above transfection reagent dropwise to the 293A cells. Change the medium 4h after transfection and replace it with 2 mL of fresh complete medium. Observe the cells. Change the medium every other day, and collect the cells when virus plaques appear (usually takes about 10 days). Centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant, add 2 mL of fresh complete medium, and place in a -80°C refrigerator. Repeat the freezing and thawing of the cell suspension at -80°C / water for 3 times, fully Vortex each time to obtain crude virus, centrifuge at 7000 rpm for 5 min at 4°C, discard the supernatant, add 2 mL of fresh complete medium, and place at -80°C for use.

[0072] Furthermore, in the above-mentioned method for preparing engineered immune cells, the virus concentration operation steps are specifically as follows: (1) Collect adenovirus-infected cells (each column is suitable for three 10 cm dishes of culture medium) (2) Balance column (4) Rinse and wash (5) Desalting and buffer exchange (6) Regeneration of purification column In another preferred embodiment, the present application also discloses a method for preparing engineered immune cells carrying both GM-CSF and functional chimeric receptor coding sequences (ie, a method for preparing CAR-M).

[0073] (1) Synthesizing the gene sequence of chimeric antigen receptor (CAR), that is, synthesizing the nucleic acid sequence of each domain of the transmembrane expression protein or polypeptide macromolecule, and cloning the coding gene sequence fragments of GM-CSF and CAR into the adenovirus expression vector Ad5F35; (2) Connecting the target fragment to a lentiviral or adenoviral overexpression vector to obtain a recombinant plasmid vector; (3) Extraction and preparation of recombinant plasmid vectors and auxiliary plasmid vectors; (4) Virus packaging and concentration; (5) Using the packaged virus to infect target immune cells, engineered immune cells are obtained that carry both GM-CSF and functional exogenous receptor coding sequences.

[0074] Furthermore, the structure of the CAR may specifically be a structure including an extracellular region, a transmembrane region and / or an intracellular domain.

[0075] More specifically, the CAR is a chimeric antigen receptor that can specifically bind to a surface protein (target) of a pathogenic cell (target cell). The engineered immune cell can achieve targeted killing of the target cell by the immune cell disclosed in the present application by expressing the artificially synthesized protein that can specifically target the corresponding target cell, thereby achieving the purpose of treating the disease.

[0076] Furthermore, the target immune cells are preferably monocytes or macrophages.

[0077] In a specific embodiment, when the target immune cells are M cells, the engineered M cells that can express both GM-CSF factor and CAR obtained based on the above steps can achieve self-secretion of GM-CSF factor, which can not only target and kill pathogenic cells, but also promote the growth and proliferation of macrophages themselves, so that the number and state of immune cells with killing ability always remain stable, that is, the effect of stable killing of target cells is achieved.

[0078] In a specific embodiment, when the target cells are tumor cells, the engineered immune cells disclosed in the present application can achieve sustained and stable killing of tumor cells.

[0079] In a specific embodiment, when the target immune cells are mononuclear cells, and further monocytes, the engineered mononuclear cells that can express both GM-CSF and CAR obtained based on the above steps are affected by the self-secreted GM-CSF factor, thereby promoting the differentiation of mononuclear cells into macrophages, and the differentiated macrophages can further maintain their own growth and proliferation, thereby further achieving the above-mentioned stable killing effect on pathogenic cells.

[0080] In a specific embodiment, when the expression vector is an adenovirus, the vector derived from the adenovirus overcomes the inherent resistance of native human macrophages to genetic manipulation and polarizes tumor cells from an immunosuppressive (M2) phenotype to a persistent pro-inflammatory (M1) phenotype, playing a positive role in the treatment of tumors.

[0081] Furthermore, based on the fact that the engineered immune cells disclosed in the present application can ensure activity, differentiation and proliferation functions, when the engineered immune cells are injected into the body to treat diseases, the single injection dose can be reduced. At the same time, the engineered immune cells disclosed in the present application can also exist in the patient's body for a long time, thereby further reducing the injection frequency of the engineered immune cells and ensuring the killing effect of the engineered immune cells on target cells.

[0082] Therapeutic applications.

[0083] The present application also discloses the use of engineered immune cells with GM-CSF sequences in the preparation of immune drugs for treating inflammatory diseases, fibrotic diseases, and solid tumor diseases to achieve the aforementioned and other purposes.

[0084] The therapeutic application of the CAR-M cells with GM-CSF gene of the present invention. The transduced M cells can target the markers of tumor cells and antibodies that specifically secrete antigens, induce immune responses of T cells and macrophages, and thus significantly improve their killing efficiency of tumor cells.

[0085] Therefore, the present invention also provides a method for stimulating an M cell-mediated immune response to a target cell group or tissue of a mammal, comprising the following steps: administering the CAR-M cells of the present invention to a mammal. In the body of a mammal, relative to CAR-M not modified by GM-CSF, the GM-CSF-modified CAR-M cells disclosed in the present application can enhance their own growth state and retention time in the body, and also achieve the effect of editing macrophages into the desired effect of being able to exert a directional killing effect, thereby achieving the maintenance of the stable killing effect of CAR-M cells.

[0086] Based on the GM-CSF-modified CAR-M disclosed in the present application, the retention time of the CAR-M in the patient's body is prolonged, and the CAR-M maintains growth and proliferation by self-secreting GM-CSF, and the injection frequency of CAR-M (or CAR-M-related drugs) during the treatment process is also reduced.

[0087] The present application also discloses a pharmaceutical composition that can continuously and stably kill engineered immune cells.

[0088] In a specific embodiment, the pharmaceutical composition includes: engineered immune cells carrying a GM-CSF coding sequence and a pharmaceutically acceptable carrier.

[0089] Furthermore, the preparation method of the engineered immune cells is as described above and will not be described in detail here. Furthermore, the engineered immune cells are mononuclear cells, M (macrophage) cells, etc. Furthermore, the engineered immune cells are preferably macrophages.

[0090] More specifically, the administration of the pharmaceutical composition can be in an autologous or non-autologous manner. For example, immune response cells and compositions comprising them can be obtained from a subject and applied to the same subject or different compatible subjects. Cells derived from peripheral blood of the disclosed subject matter of the present invention or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered by including catheter administration, intravenous injection or parenteral administration. When the pharmaceutical composition of the disclosed subject matter of the present invention is administered, it is usually formulated into a unit dose injectable form (solution, suspension, emulsion).

[0091] The composition of the present application can be a preparation. The immune response cells of the chimeric antigen receptor (CAR) expressing the specific targeting target cells disclosed in the present application and the composition comprising the same can be conveniently provided as a sterile liquid preparation, such as an isotonic aqueous solution, a suspension, an emulsion, a dispersion or a viscous composition, which can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions and solid compositions. In addition, liquid compositions are more convenient to apply, particularly by injection.

[0092] On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide longer contact time with a particular tissue. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium including, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0093] Various additives may be added to enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers.

[0094] According to the pharmaceutical compositions disclosed in the present application, any carrier, diluent or additive used must be compatible with the engineered immune cells carrying the GM-CSF coding sequence of the presently disclosed subject matter.

[0095] If necessary, the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent. The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, such as a liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel or another liquid form, such as a time-release form or a liquid-filled form).

[0096] Example 2: Specific steps for preparing the aforementioned CAR-M.

[0097] When the target immune cells are macrophages and the expression vector is an adenovirus vector, the preparation of engineered immune cells with GM-CSF coding sequences is faster and more efficient. The experimental steps are as follows: S1 Plasmid construction of Ad5F35-CAR-PGK-GM-CSF: The gene fragments of CAR and GM-CSF are synthesized, and the GM-CSF coding gene is shown in SEQ ID NO. 1. The GM-CSF coding gene sequence fragment is connected to the adenovirus expression vector Ad5F35 to obtain the plasmid Ad5F35-CAR-PGK-GM-CSF that can simultaneously express the target CAR and the GM-CSF that enhances the expression of CAR.

[0098] Plasmid map Figure 2 As shown, the vector Ad5F35-CAR-PGK-GM-CSF uses the EF1a promoter to drive the expression of the CAR sequence; the CAR sequence is obtained by sequentially connecting the extracellular region, the transmembrane region and the intracellular region sequence fragments; and the vector uses the PGK promoter to drive the expression of the GM-CSF sequence.

[0099] Construction of S2 chimeric antigen receptor macrophage (CAR M) cells Main process of CAR-M construction and CAR expression: S21. Plasmid synthesis S22. Adenovirus packaging and concentration S23, adenovirus infection of primary human macrophages S24. Detection of target antigen receptor expression 1. Use the above construction method to construct a CAR plasmid that can target a specific target.

[0100] 2. Adenovirus packaging.

[0101] Prepare linearized Ad5F35 plasmid and 293A cells in advance. 24 h before transfection, plate 293A cells to 35 mm cell culture dishes, with a confluency of 80-90% and good condition. Transfect plasmids as follows: Take jetPRIME out of the 4°C refrigerator, restore to room temperature, and shake well before use. Take 4 μg of the linearized plasmid constructed in Example 1, add 200 μL PRIMEBuffer, and vortex for 10 seconds; then add 8 μL PRIME, vortex for 10 seconds, let stand at room temperature for 10 minutes, and then add the above transfection reagent dropwise to 293A cells. Change the medium 4 hours after transfection with 2 mL of fresh complete medium. Observe the cells. Change the medium every other day, and when virus plaques appear (usually takes about 10 days), collect the cells. Centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant, add 2 mL of fresh complete medium, and place in a -80°C refrigerator. The above cell suspension was repeatedly frozen and thawed at -80℃ / water for 3 times, and fully vortexed each time to obtain crude virus. The crude virus was centrifuged at 7000 rpm at 4℃ for 5 min, the supernatant was discarded, 2 mL of fresh complete culture medium was added, and the suspension was placed at -80℃ for use.

[0102] S3. Concentration of virus fluid.

[0103] The specific steps are as follows: (1) Collect adenovirus-infected cells (each column is suitable for three 10 cm dishes of culture medium) Transfer 10 mL of culture supernatant to a new 15 mL Centrifugal Tube, leaving 5 mL of supernatant. Collect the cells in the culture dish with a scraper, transfer the cells and supernatant to another new 15 mL CentrifugalTube, freeze in a dry ice / ethanol mixture, and thaw at 37°C, repeat 3 times, and mix the lysate and 10 mL of supernatant. Centrifuge at 4°C, 3,000 rpm for 10 min, collect the supernatant, and filter with a 0.45 μm filter. The filtered supernatant can be used for purification or stored at -80°C for later use.

[0104] (2) Balance column Dilute 10× AV Wash Buffer to 1× AV Wash Buffer with ddH2O; dilute 2× AV Elution Buffer to 1× AV Elution Buffer with ddH2O.

[0105] Place the AV Mini Column in a 15 mL Centrifugal Tube and centrifuge at 4°C, 500 × g for 2 min. Fix the AV Mini Column with a clamp or other bracket, break off the tip at the bottom, loosen the cap, and let the liquid drain from the AV Mini Column under gravity. Once the liquid stops dripping, slowly add 2 mL ddH2O, wait for the solution to flow out of the AV Mini Column, then add 5 mL 1× AV Wash Buffer and continue to let the solution flow away.

[0106] (3) Upper column Transfer 5 mL of supernatant to the AV Mini Column and allow it to flow through by gravity. Continue adding supernatant to the AV Mini Column until all the supernatant has passed through.

[0107] (4) Rinse and wash Add 5 mL of 1× AV Wash Buffer to the AV Mini Column and repeat once. Centrifuge by gravity flow or at 1,000 × g for 5 min at 4°C.

[0108] Add 4 mL of 1× AV Elution Buffer to elute the virus and collect 4 ml of the eluate.

[0109] (5) Desalting and buffer exchange Transfer 4 mL of the sample collected in the previous step to the Centrifugal Filter and centrifuge at 4°C, 3,000 rpm for 10-15 min until about 500 μL of solution remains in the Centrifugal Filter. Discard the filtrate and add 3.5 mL of PBS to the Centrifugal Filter. Centrifuge at 4°C, 3,000 rpm for 10-15 min until about 400-500 μL of solution remains in the Centrifugal Filter. Pipette up and down several times in the Centrifugal Filter and transfer the virus-containing solution to a clean tube.

[0110] (6) Regeneration of purification column After purification, add 5 mL of Regeneration Buffer to the column, and let the solution flow through the column by gravity. Add 5 mL of 1× AV Wash Buffer, cover the bottom cap tightly, seal the column with sealing film, seal it in a bag, and store it at 4℃ for later use.

[0111] S4. Adenovirus infection of primary human macrophages.

[0112] Virus concentrate was added to primary human macrophages at a ratio of 2 (MOI of total virus pfu to total cell number) during infection, mixed and incubated in an incubator containing 5% CO2, and T cell complete medium was added 4 hours later to adjust the cell density to 1.0×106 / mL for culture transduction to obtain CAR M cells. CAR M can be induced into CAR M1 by adding 50ng / ml PMA for 24h, and then adding 20ng / ml IFNγ and 500pg LPS for 24h.

[0113] CAR M induced by IFNγ and LPS is characterized by an increase in M1 markers and a decrease in M2 markers. It can accurately express CAR and has target effector activity, targeting and phagocytizing tumor cells that highly express Her2.

[0114] S5. Detection of CAR expression by WB.

[0115] ① Cell lysis ② Protein quantification ③ SDS-PAGE gel electrophoresis (1) Gel preparation: Use One-Step PAGE Gel Fast Preparation Kit (10%); for homemade gel, see Appendix - SDS-PAGE gel preparation protocol.

[0116] (2) Sample preparation: Add 5×Laemmli buffer (5×Laemmli buffer: 0.15 M Tris-HCl pH 6.8, 5%SDS, 25% Glycerol, and 0.05% Bromophenol blue) containing 3% β-mercaptoethanol and 200 mM DTT (β-mercaptoethanol and DTT are added immediately before use) to the cell lysate, incubate at 95°C in a metal bath for 8 min, centrifuge at 12,000 rpm for 2 min, and use the supernatant for sample loading.

[0117] (3) Sample loading (4) Electrophoresis: 80 V constant voltage electrophoresis.

[0118] ④ Transfer (1) PVDF membrane activation: Place the membrane in methanol and soak for 5 min.

[0119] (2) Wet transfer conditions: 80 mA transfer for 180 min.

[0120] ⑤ Closed Place the PVDF membrane in blocking solution (5% unsweetened soy milk / skim milk powder using TBST as solvent) and incubate on a shaker at room temperature for 1-2 h.

[0121] ⑥ Primary antibody labeling Incubate overnight on a shaker at 4°C. Dilute the primary antibody with blocking buffer at a dilution ratio of 1:1000.

[0122] ⑦ Washing Incubate with TBST on a shaker at room temperature for 10 min each time, for a total of 3 times.

[0123] ⑧ Secondary antibody labeling Incubate on a shaker at room temperature for 1 h, and dilute the secondary antibody with blocking buffer at a dilution ratio of 1:2000.

[0124] ⑨ Washing Incubate with TBST on a shaker at room temperature for 10 min each time, for a total of 3 times.

[0125] ⑩ Imaging.

[0126] The data of CAR-M secreting GM-CSF factor obtained by infecting macrophages with adenovirus at different MOIs are shown in the figure. Figure 3 shown.

[0127] Wherein, M is a macrophage, CAR-M is a cell inoculated with adenovirus in M ​​cells, MOI is the multiplicity of infection, which refers to the number of viruses infected in each cell, the culture medium is the negative blank control, and the culture medium with 10 ng / mL of GM-CSF added is the positive control. It can be seen that based on the technical solution disclosed in the present application, the MOI value can be adjusted according to the concentration of GM-CSF factor secreted as needed.

[0128] Comparison of macrophage recovery between GM-CSF and M-CSF.

[0129] Human monocyte-derived macrophages were cultured in complete medium containing GM-CSF (10 ng / mL) and M-CSF (100 ng / mL) for 7 days, and the macrophage recovery rate was calculated. Figure 4 As shown, the horizontal axis represents different seeding densities, and the vertical axis represents the recovery rate of macrophages (the amount of macrophages obtained / the amount of monocytes seeded × 100%). It can be seen that under different seeding densities, GM-CSF (10 ng / mL) can obtain a higher recovery rate of macrophages.

[0130] Comparison of macrophage activity of GM-CSF and M-CSF.

[0131] Human monocyte-derived macrophages were cultured in complete medium containing GM-CSF (10 ng / mL) and M-CSF (100 ng / mL) for 7 days, and the activity of macrophages was compared. The results of flow cytometric analysis of GM-CSF and M-CSF are shown in Figure 2. Figure 5A-5B As shown, Figure 5A The cell death rate of macrophages cultured with M-CSF factor was 7.68, of which Figure 5B When macrophages were cultured with GM-CSF, the cell death rate was 1.20. It can be seen that the macrophage activity obtained by the medium containing GM-CSF (10 ng / mL) was higher.

[0132] It can be seen that the CAR-M prepared based on the method disclosed in the present application for continuously and effectively killing macrophages can be achieved by controlling the MOI value according to the required GM-CSF factor concentration of the cultured macrophages, thereby obtaining a state that enables the CAR-M to always maintain a stable killing state.

[0133] Comparison of in vivo retention data of CAR-M cells (with GM-CSF) and M injected in different ways.

[0134] Figures 6A-6D These are experimental data of CAR-M and macrophages prepared by the preparation method disclosed in this application, which were injected into mice and observed for 13 days.

[0135] Tail vein injection.

[0136] The lipophilic fluorescent dye DiR iodide was used to fluorescently label CAR-M cells (with GM-CSF) and macrophages in vitro, making the cells emit near-infrared fluorescence. Ten million fluorescently labeled cells of each type were injected into the tail vein of tumor-bearing nude mice, and the mice were imaged in vivo at different time points. The fluorescence intensity gradually decreased with cell death. Fluorescence imaging of mice injected with CAR-M cells (with GM-CSF) and macrophages in the tail vein is shown in Figure 2. Fig. 6A shown.

[0137] The fluorescence intensity of in vitro imaging of each group of mice at each time point was quantified, and the average value of the total fluorescence intensity of each group of mice was taken to draw a scatter plot and trend line. Figure 6BAs shown, the solid trend line is the macrophage group, the dotted trend line is the CAR-M group, the slope of the macrophage group is: -6E+08, and the slope of the CAR-M group is: -2E+08. It can be seen that the absolute value of the slope of the CAR-M group is smaller than that of the macrophage group, that is, the fluorescence decrease rate of the CAR-M cell group is lower than that of the ordinary macrophage group, indicating that through the tail vein injection, the death rate of the CAR-M cells with GM-CSF disclosed in the present application in mice is lower than that of macrophages, thereby achieving the effect of stable existence of CAR-M cells in mice.

[0138] Intratumoral injection.

[0139] The lipophilic fluorescent dye DiR iodide was used to fluorescently label the CAR-M cells (with GM-CSF) and macrophages prepared by the present invention in vitro, so that the cells emitted near-infrared fluorescence. Ten million fluorescently labeled cells of the two types were injected into tumor-bearing nude mice, and the mice were imaged in vivo at different time points. The fluorescence intensity gradually decreased with the death of cells in the mice. Fluorescence imaging of mice injected with CAR-M cells (with GM-CSF) and macrophages in the tumor is shown in Figure 2. Figure 6C shown.

[0140] The fluorescence intensity of in vitro imaging of each group of mice at each time point was quantified, and the average value of the total fluorescence intensity of each group of mice was taken to draw a scatter plot and trend line. Fig.6D As shown, the solid trend line is the macrophage group, the dotted trend line is the CAR-M group, the slope of the macrophage group is: -2E+09, and the slope of the CAR-M group is: -1E+09. It can be seen that the absolute value of the slope of the CAR-M group is smaller than that of the macrophage group, that is, the fluorescence decrease rate of the CAR-M cell group is lower than that of the ordinary macrophage group, indicating that the CAR-M cells with GM-CSF prepared by the preparation method disclosed in the present application through intratumoral injection have a lower death rate in mice than macrophages, thereby achieving the effect of stable existence of CAR-M cells in mice.

[0141] In summary, the method for preparing macrophages disclosed in the present application, regardless of the mode of administration, can achieve the extension of the retention time of CAR-M in mice, and achieve the continuous and stable killing effect of CAR-M on tumor cells in vivo. The corresponding structures, actions, behaviors, and equivalent forms of all device elements or step elements disclosed in the present application are intended to include any structure, action, and behavior for performing functions in combination with other components claimed for protection as specifically requested. Descriptions of various preferred embodiments of the present disclosure are given for the purpose of disclosure, tending to cover various variations and equivalent arrangements, rather than as an exhaustive list or limitation. The terms used here are selected to best explain the principles of the embodiments, the practical applications or technical improvements that are superior to those found in the market, and the purpose of the present invention has been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and illustrated in the embodiments, and the embodiments of the present invention may be deformed or modified without departing from the principles.

Claims

1. A method for enabling macrophages to kill cells continuously and effectively. It is characterized in that The method comprises: Constructing a vector including a target gene fragment, wherein the vector is a viral vector or a non-viral vector; Prepare target immune cells based on the vector to obtain engineered immune cells; Wherein said constructing a vector including the target gene fragment comprises encoding the gene sequence of the exogenous GM-CSF protein into said vector; The viral vector is an adenovirus or a lentivirus.

2. The method for causing macrophages to be continuously and effectively killed according to claim 1, It is characterized in that The target gene fragment also includes a gene sequence encoding a functional chimeric receptor.

3. The method for continuously and effectively killing macrophages according to claim 2, It is characterized in that The macrophages are engineered macrophages.

4. The method for causing macrophages to be continuously and effectively killed according to claim 1, It is characterized in that The target immune cell is a macrophage or an immune cell that can differentiate into a macrophage.

5. An expression vector, It is characterized in that The expression vector is prepared by the method of claims 1-3, wherein the expression vector comprises a gene sequence encoding an exogenous GM-CSF protein, and wherein the expression vector is an adenovirus expression vector or a lentivirus expression vector.

6. A GM-CSF engineered immune cell, It is characterized in that The GM-CSF engineered immune cells are prepared by the preparation method of the GM-CSF engineered immune cells according to claims 1-4.

7. Application of GM-CSF engineered immune cells, It is characterized in that Application of the GM-CSF engineered immune cells in treating inflammatory diseases, fibrotic diseases, and solid tumor disease immune cell drugs; The engineered immune cells are the engineered immune cells prepared according to claims 1-4.

8. A pharmaceutical composition, It is characterized in that The pharmaceutical composition comprises the GM-CSF engineered immune cells disclosed in claim 6 and a pharmaceutically acceptable carrier.

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